Pharmacy Courses

Interview Questions and Answers on Chromatography (Part-1)

What do you mean by analytical method development?

Analytical method development is a procees to develop a method which is suitable for analysis to establish the identity, purity, physical characteristics, and potency of drugs, including the drug's bioavailability and stability.

What do you mean by Analytical method validation?

Method validation is the process that is used to verify the analytical process use of a particular method, an analytical instrument is proper for its intended use. Method validation results are used to judge the quality, quantity, consistency, and reliability of particular results. 

Method validation in chromatography must be carried out as per the validation protocol is given by the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) or The United States Pharmacopeia (USP) and British Pharmacopoeia (BP). The protocol includes the process and criteria of acceptance for all protocol.

As per ICH guidelines, below listed are the method validation parameters of pharmaceutical analysis.

  • Accuracy
  • Precision
  • Specificity
  • Limit of Detection (LOD)
  • Limit of Quantitation (LOQ)
  • Linearity
  • Range
  • Robustness
  • Rougdness
  • System Suitability

What do you mean by Accuracy?

Accuracy is a measure of the closeness of the experimental value to the standard. 

It's a method validation parameter. The reference standard is a preferred technique by direct comparison accuracy. In this technique of validation parameter, perform recovery study by selecting three different concentrations like 0 % (0 % is your standard) 80 %, 100 % and 120 %. Compare to a standard sample and recovery should be in the range of 99 to 101 %.

What do you mean by Precision?

Precision is the degree of agreement among individual test results when the procedure is applied repeatedly to multiple samplings.

There are three parameters in the precision study: Repeatability, Intraday Precision, and Interday Precision. In the repeatability study spikes the same sample solution six times and the result should have RSD ≤ 1.0 %.

In intraday precision inject the sample of three different concentrations and should have RSD ≤ 2 %. And In Interday inject the same sample of three different concentrations within 2 to 3 days at the same conditions and should have RSD ≤ 2 %.

What do you mean by Specificity?

Specificity is the ability of the analytical method to distinguish between the analyte(s) and the other components in the sample matrix.

Specificity is a method validation parameter, Spike your sample in addition with degradation products, solvents, chemicals and solvents used to perform analysis, hence all analytes should be separate and minimum resolution have 2.0. and another one is to spike samples of marketed preparation, it should be separate having RSD ≤ 2 %.

What do you mean by Limit of Detection (LOD)?

LOD is a lower limit of detection, the lowest amount of an analyte can be constantly recognized with a known analytical method. S/N’ ratio > 3.

What do you mean by Limit of Quantitation (LOQ)?

Limit of Quantitation (LOQ) is the lowest concentration of a component in a sample which can be determined by a known analytical method and S/N’ ratio > 10.

What do you mean by Linearity?

Linearity is important for the confirmation of the method's sensitivity for the analysis of the analyte's concentration within a defined range.

In this parameter of method validation, the range of 0-150% of the expected level of analysis should be covered. The method should display linearity in the desired range. For the linearity purpose, prepare six samples in desire range to take linearity and correlation coefficient value should be minimum R² = 0.99.

What do you mean by Robustness?

The robustness of an analytical procedure is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters.

The rigid method is that which tolerates the slight diversity in the situation of experimental conditions and can easily run by the average chromatographer on a different system at different places and should not necessary to use the same system to apply the method.

What do you mean by Retention Time (tR)?

Retention time is the time required by the analyte from injection to migrate from the column to the detector.

What do you mean by system suitability?

System suitability is to prove that system is working perfectly before the analysis on HPLC, GC, TOC analyzer or any other system. It is required to done before every sample analysis.

What do you mean by Resolution (Rs) in HPLC?

Resolution is the measurement of the separation of two peaks of different retention time in a chromatogram.

In chromatography analysis, if the sample having more than one compounds then need to be separate properly. If the retention time of two peaks have adequately different or peaks are sharp then get better resolution. The resolution shows the separation and sharpness of peak, an acceptable limit of resolution are Rs >1.5.

Rs = 2(t2-t1)/w1+w2

What do you mean by Capacity Factor (k')?

Capacity factor (k') is a signal of how extended an analyte can be retained over the stationary phase. Or analyte has how much interact with the stationary phase. Capacity factor (k') depends as mobile phase, stationary phase, quality of column and temperature.

For better chromatographic performance in isocratic separation capacity factor (k') must be in between 1-10 k'

Capacity factor calculated as, k'= (Tr - To) / To.

What do you mean by Theoretical Plates (n) in chromatography?

The theoretical plate number is a significant factor of the column; it is work as tool of column efficiency measurement. 

Its illustrate the quality of separation and ability to generate narrow and sharp peaks. Column having more number of theoretical pates are consider as more efficient in separation than the plates with less number, hence the peak resolution and narrow peak are depend upon the column efficiency.

What is column chromatography? What are the Advantages and Disadvantages of Column Chromatography?

Column chromatography is a general technique that is used to separate the compounds from the complex mixtures. It can use small or large-scale column chromatography to separate and purify the analytes. Column chromatography has two phases, a mobile phase (liquid) and a stationary phase (solid). The sample mixture of compounds travels with the mobile phase through the stationary phase and basis of different degrees of adhesion it separates.The column chromatography works on the principle of adsorption. There are four different column chromatography types for several applications and that work on different mechanisms.

Advantages of column chromatography:

  • Using column chromatography all kinds of complex mixtures can be separated.
  • Any amount of mixture can be separated by column chromatography.
  • A broad range of mobile phases.
  • Analytes can be separated and reused, in preparative type chromatography.
  • It can be possible to run automation.
  • This is a robust method.

Disadvantages of column chromatography:

  • It takes more time to separate the compounds.
  • Column chromatography has of low separation power relative to advanced separation techniques.
  • Higher quantities of solvents are essential, which is more expensive.
  • Automation makes more complex and costly.

What are the Advantages and Disadvantages of Gas Chromatography?

Gas chromatography (GC) is the significant analytical method for the separation of volatile compounds in a mixture by injecting a liquid or gaseous sample into a mobile phase, usually called the carrier gas, and passing the gas through a stationary phase. The mobile phase consists of an inert gas it may be nitrogen, helium, or argon and the column can be packed or capillary they can available in different diameters and lengths as per the requirement of the sample.


Advantages of gas chromatography (GC):
  • The major advantage of gas chromatography is its high sensitivity, resolution, and separation ability, which allows it to separate a wide range of volatile compounds.
  • It can be upgraded to a mass spectrometer (MS), which is used to determine the mass-to-charge ratio of ions.
  • It comes with a variety of detectors and injectors that can be used for various pharmaceuticals as well as other applications.
  • Gas chromatography can analyze a sample much faster than other chromatographic techniques.
  • It is a robust method of separation that gives the superior signal-to-noise ratio.
  • It only takes a very small amount of sample to inject, and its detectors are extremely sensitive, allowing it to detect extremely low concentrations (ng-pg).
  • As per the requirement of the molecule, there are different types of GC columns are available in many diameters and lengths.
  • Gas chromatography is easy, automated, and has quick analysis of data which gives comparatively high precision, accuracy, and reproducible results.
  • Operational parameters such as flow rate, temperature, and pressure, etc. are easy to change even during chromatographic runs.

Disadvantages of a gas chromatography (GC):
  • The major disadvantage of GC is that only volatile and thermally stable compounds can be separated using gas chromatography.
  • Detectors which are used in the GC are destructive, except for MS.
  • Selectivity in HPLC or TLC is also better as a mobile phase can be easily changed. In GC, you can just modify the temperature of the column and oven, but you cannot change the mobile phase as it has a constant flow of carrier gas (helium, nitrogen).
  • Since hydrogen gas, which is used for flame, is highly flammable, care must be taken when using it.
  • It is impossible to recover individual sample components.

What are the advantages and disadvantages of HPLC?

The advantage of HPLC:

  • HPLC offers a rapid, automated and highly precise method to recognize certain chemical components in a sample.
  • High-performance liquid chromatography offers a fast and precise quantitative analysis.
  • A gradient solvent system can be applied in certain methods.
  • It is highly reproducible.
  • HPLC can be upgraded to mass spectroscopy (MS).
  • The HPLC is very rapid, efficient, and delivers high resolution as compared to other chromatographic techniques, such as TLC, column chromatography, and paper chromatography.
  • Manages all areas of analysis to increase productivity.

The disadvantage of HPLC:

  • HPLC can be an expensive method, it required a large number of expensive organics, needs a power supply, and regular maintenance is required.
  • It can be complicated to troubleshoot problems or develop new methods.
  • The lack of a universal detector for HPLC, however, the UV-Vis detector only detects chromophoric compounds.
  • The separation in High-performance liquid chromatography has less efficiency than GC.
  • It is more difficult for the beginner.
  • HPLC pump process reliability relies on of cleanliness of the sample, mobile phase, and proper operation of the system.


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