Chromatography Interview Questions & Answers

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Chromatography Interview Questions & Answers

Looking for an excellent career in Chromatography? Are you confused about the interview questions? Then wisdomjobs site will help you to explore all the options and build up an excellent career in Chromatography. Chromatography is a process where a chemical mixture carried by a gas or liquid is divided into components as an outcome of discrepancy distribution of the solutes as they run around or over a fixed solid or liquid phase. There are several Chromatography jobs available in the market in different positions like Research Associate, QC Executive, QC Chemist, Scientist, Lecturer, Research Scholar, Microbiologist, Research Assistant etc. Please visit our Chromatography job Interview questions and answers page designed by our experts that will help you to grab attention by the hiring recruiter and makes your job search process easier.

Chromatography Interview Questions

Chromatography Interview Questions
    1. Question 1. What Are The Main Differences Between High Performance Liquid Chromatography And Gas Chromatography?

      Answer :

      • In HPLC the mobile phase is a liquid whereas in Gas Chromatography the mobile phase or carrier is a gas.
      • HPLC is useful for analysis of samples which are liable to decompose at higher temperatures. GC involves high temperatures so compounds are stable at such temperatures.
      • Gas Chromatography is applied for analysis of volatile compounds whereas non volatile compounds can be easily analyzed on HPLC
      • Gas Chromatography cannot be used for analysis of high molecular weight molecules whereas HPLC has applications for separation and identification of very high molecular weight compounds
      • HPLC requires higher operating pressures than GC because liquids require higher pressures than gases for transport through the system
      • HPLC columns are short and wide in comparison to GC columns

    2. Question 2. Which Type Of Gc Detector Is Most Commonly Used? Explain Its Working Principle And What Are Its Limitations?

      Answer :

      The most commonly used detector is the flame ionize detector. The sample is combusted with the help of fuel gas and oxidant in the detector body. Combustible sample components burn and produce ions and electrons which can conduct electricity through the flame. A large potential difference is applied at the burner tip and the collector electrode located above the flame and the current between the electrodes is measured. The detector is mass sensitive and response is not affected by carrier gas flow rate changes. However, the detector is not responsive to inorganic gases such as CO, O2, NH3, N2, CS2, CO2, etc.

    3. Question 3. What Are The Commonly Used Carrier Gases In Gc Analysis When Using Fid Detector?

      Answer :

      Inert gases commonly used in analysis when using FID detector are Nitrogen and Helium. Nitrogen is more commonly used as it is less expensive than Helium. Purity of carrier gas should be more than 99.995% and on-line traps should be used to prevent residual moisture or other impurities from entering the system.

    4. Question 4. What Are The Desirable Characteristics Of A Gc Detector ?

      Answer :

      The detector chosen for particular analysis should :

      • Give reproducible response to changes in concentration of eluting compounds in the carrier gas stream.
      • Should provide a large linear dynamic range 
      • Should have high sensitivity 
      • Should have small internal volume to give narrow peaks and also facilitate flushing of previous sample traces 
      • Should preferably be non-destructive

    5. Question 5. What Do You Understand By Specificity Of A Detector?

      Answer :

      Detectors falls into three categories depending upon response to the eluting compounds:

      • Non-selective – Respond to all component in the gas stream except for the carrier gas
      • Selective – Respond to a particular class of compounds with common physical or chemical properties 
      • Specific – Respond to a single specific compound only in the carrier gas stream

    6. Question 6. What Are The Commonly Used Types Of Capillary Columns?

      Answer :

      Capillary columns are generally 10 – 100m long tubes having an internal diameter ranging from 0.1 – 0.5mm made of flexible material such as fused silica. Common types of capillary columns are

      • Wall coated open tubular (WCOT) – Internal wall is coated with a very fine film of adsorbing liquid 
      • Surface coated open tubular (SCOT) – Inner wall is lined with a layer of solid support on to which the liquid phase is absorbed. 

      The columns are flexible and wound into several turn coils supported on a SS cage inside the column oven

    7. Question 7. What Do You Understand By Column Efficiency And How It Is Expressed?

      Answer :

      On continuous use a column gradually loses its original resolution power. Column efficiency is expressed on the basis of plate theory concept. Each component under separation spends a finite time in each theoretical plate. Smaller the plate height the larger the number of plates (N) and better is the column efficiency.

    8. Question 8. What Do You Understand By Temperature Programming In Gc Analysis?

      Answer :

      Temperature programming means change of temperature of the column at a rate predetermined rate during the analytical run. This has the same influence on elution time of separated components as gradient programming in HPLC analysis. Temperature programming helps reduce analysis time by permitting early elution of less volatile components.

    9. Question 9. When Is Isothermal Operation Useful?

      Answer :

      Isothermal operation is useful when high resolution is required for separating compounds having narrow boiling range. Temperature is set to around mid range of boiling points of constituents. This results in good resolution of low boiling components but band broadening of higher boiling components can result due to their longer retention in the column.

    10. Question 10. What Measures You Would Adopt To Extend Useful Life Of A Column?

      Answer :

      • Condition a column before first use or after long time storage 
      • Take care not to exceed upper temperature limit specified by the manufacturer 
      • Avoid injection of solutions which are strongly acidic or basic in nature 
      • Rinse columns by injection with blank solvents such as methanol, methylene chloride or hexane to remove contamination of column after excessive usage

    11. Question 11. What Is The Basic Principle Of Paper Chromatography?

      Answer :

      Paper chromatography is a form of liquid chromatography where the components of a mixture of organic compounds get separated as unique spots by unidirectional flow of the developing liquid mobile phase solvent mixture over the filter paper to which a spot of the sample is applied. The distance travelled by each component is specific under the given set of operational conditions.

    12. Question 12. Why The Developing Solvent Mixture Is Prepared Fresh Before Use?

      Answer :

      The developing liquid phase comprises of a pure solvent but more often it is a mixture of two or more solvents in specified proportions. In case solvents are mixed and stored for long periods there could be loss of volatile component which will alter the mixing proportions.

    13. Question 13. Why Is It Necessary To Cover The Developing Chamber During The Paper Development?

      Answer :

      During the chromatogram development chamber is covered.This is essential as the environment inside the chamber should remain saturated with the solvent vapour. Development times can vary from about an hour to several hours and a saturated environment prevents losses due to evaporation.

    14. Question 14. What Are The Common Techniques Used For Detecting Colourless Spots?

      Answer :

      It is easy to distinguish coloured spots visually but for colourless compounds alternate techniques need to be adopted which can be specific or non-specific.

      • A common non-specific method is suspension of developed chromatogram in iodine vapour. Most organic compounds show up as brown spots.
      • The sheet is viewed in a UV Viewing cabinet under 366 nm and 254 nm wavelength lamp illumination. On observation the spots need to be carefully marked with a pencil for Rf calculations.
      • Under specific methods amines and amino acids are observed by spraying heated paper on development with 0.2% hydrazine. Deep blue or purple spots begin to appear.
      • Alkaloids – Dragendroff’s reagent spray results in orange or orange yellow spots.
      • Aldehydes&Ketones – 2,4-DNPH spray in methanol and sulphuric acid results in orange or yellow spots.

    15. Question 15. Why Should The Samples Have Reasonable Solubility Which Is Neither Too High Or Too Low In The Developing Solvent Mixture?

      Answer :

      The samples should have a medium solubility in the developing solvent mixture.Too high a solubility will lead to transfer of the component alongwith the solvent front and on the other hand if the solubility is too low the component will not be carried by the solvent mixture and will remain close to the initial applied spot. In either case the resolution of the mixture components will be low. Thus reasonably good resolution can be obtained for medium solubility of compounds in the solvent mixture.

    16. Question 16. What Information You Get From The Retardation Factor Value?

      Answer :

      Retardation factor Rf is a measure of the separation of a particular component. It is expressed as

      Rf = distance moved by the component spot/ distance moved by solvent front

      Rf is a unit less quantity and lies between 0and 1.A value of 0 indicates no separation has taken place and 1 represents that the component has moved entire length alongwith the solvent front. In case two spots have same value of Rf it indicates that they are not resolved. At least a difference of 0.05 is necessary to discern the separation between two spots.

    17. Question 17. Can You Remember The Various Paper Chromatography Techniques?

      Answer :

      Paper chromatography separations are classified in accordance with the direction of flow of mobile phase along the filter paper.

      • Ascending paper chromatography – the carrier liquid moves from bottom upwards.
      • Descending paper chromatography – the carrier liquid trough is on top and mobile phase moves downward on the filter paper.
      • Ascending – descending paper chromatography – The paper is rolled downward over the rod at the top. On reaching the top in ascending mode it starts downward movement in the next phase.

    18. Question 18. What Are Essential Criteria For Selection Of Suitable Solvents For Paper Chromatography?

      Answer :

      Solvents are selected on the basis of solubility of the sample components. In general it is advisable to keep in mind:

      • Solvents are not toxic or carcinogenic.
      • Solvent constituents of mixture should not react with any of the sample constituents.
      • Solvents selected should not interfere in detection of separated spots.
      • Solvents should not be highly volatile as loss of components can result in change of mixture composition.

    19. Question 19. Why Paper Chromatography Has Retained Its Applicability In The Face Of A Emergence Of Advanced Instrumental Techniques?

      Answer :

      Chromatographic technique of analysis has seen an impressive growth over time. Such advances have increased laboratory throughputs lowered limits of detection and has made forays into new areas of applications. Paper chromatography has retained its ground till date and is popular in laboratories across the world. Some of the reasons for this are:

      • Low cost of analysis and freedom from maintenance.
      • Separated spots are visible for coloured compounds and colourless compounds can be viewed by using alternate techniques.
      • Minimum operation and training requirements.Solvent consumption is much less as compared to more sophisticated techniques.
      • Paper chromatography serves as a good demonstration of basic concepts of separation for school and undergraduate students.

    20. Question 20. What Are The Limitations Of Paper Chromatography Technique?

      Answer :

      Paper chromatography has some limitations such as:

      • Semi-quantitative in nature.
      • Overlapping of spots of components having close Rf values.
      • Higher concentration of components often leads to streaking instead of well-defined spots.
      • Errors in Rf calculations can result from uneven flow of solvent front. This can be caused by running out of solvent at the bottom of the chamber, uneven cutting of the filter paper or unevenness of the bottom of the development chamber.
      • Improper sample spotting, spotting below the marked line resulting in dipping into the solvent or accidental dipping of spot into solvent while inserting the paper into the solvent chamber.

    21. Question 21. Which Type Of High Performance Liquid Chromatography Technique Is Most Widely Used?

      Answer :

      Reverse phase Chromatography has the widest range of applications. The stationary phase comprises non polar  organic chains bound to inert silica surface and mobile phase comprises of aqueous or aqueous-organic mixtures  comprising of polar solvents of varying degrees of polarity. The elution sequence is polar followed by less polar and least polar or non polar compounds eluting last through the column.

    22. Question 22. What Is The Separation Principle In Size Exclusion Chromatography?

      Answer :

      In size exclusion chromatography the separation does not involve chemical interactions between eluting molecules and stationary phase. The separation takes place on the basis of molecular size with larger molecules eluting first and small molecules in the end. Small molecules are retained longer in the pores of the stationary phase therefore they get eluted last.

    23. Question 23. Why Is It Necessary To Degass The Mobile Phase?

      Answer :

      Mobile phases entrap air from the atmosphere and this trapped air gets released as small bubbles under high pressures encountered during the HPLC analysis. Such bubbles can lead to noise in detector response or hinder flow of mobile phase through columns. In order to overcome such problems degassing of mobile phase becomes essential.

    24. Question 24. Which Is The Most Commonly Used Detector In High Performance Liquid Chromatography And Why?

      Answer :

      The most commonly used detector in HPLC is the UV-VIS detector. The reason for its predominant use is that it gives specific response to a particular compound or class of compounds. Most of the organic compounds absorb at specific wavelengths covered in the available wavelength range of the detector.

    25. Question 25. What Do You Understand By A Bulk Property Detector And A Specific Property Detector?

      Answer :

      A bulk property detector responds to some property of mobile phase and sample combination passing through it at any point of time such a Refractive index or Electrochemical detector whereas a specific property detector is responsive only to the characteristic property of the eluting molecule and is independent of changes in mobile phase composition such as UV-Vis and Fluorescence detectors.

    26. Question 26. What Do You Understand By Isocratic And Gradient Elution?

      Answer :

      When the composition of the mobile phase is not changed through the chromatographic  run  the operation is termed as isocratic. It can involve a single solvent or a mixture of two or more solvents mixed in a fixed proportion. In gradient operation the composition at start of run is programmed to change at a predetermined rate and the composition at the end of run is different from the composition at the start.

    27. Question 27. What Are The Desirable Features Of A High Performance Liquid Chromatography Detector?

      Answer :

      The desirable features of a detector are

      • Sensitivity towards solute over mobile phase.
      • Low  dead volume to eliminate  memory effects
      • Low noise
      • Low detection limits
      • Large dynamic linear range

    28. Question 28. What Do You Understand By Theoretical Plate Concept And How Hetp Affects The Separation Of Hplc Column?

      Answer :

      Plate theory concept was introduced to explain efficiency of columns. The concept assumes that a state of instantaneous equilibrium exists    between the concentration of solute in stationary phase and the mobile phase and further the column is imagined to be divided into a number of theoretical plates. Any analyte spends a finite time in each plate and this is the equilibrium time. Smaller the plate height the greater is the number of plates in a given length (HETP) and better is the column resolution.

    29. Question 29. What Are The Benefits Of Fast Lc Or Uhplc?

      Answer :

      Fast or UHPLC technique makes use of small particles below 2 μ size Use of such particle sizes result in high resolution and as small columns can be used  it results in completion of analysis in much less time thereby reducing consumption of expensive solvents.

      We hope you had a great learning experience through the introductory free e-learning HPLC course. I shall remain in contact with you for our offerings on advance versions of the HPLC e-learning courses and subsequent introduction covering other analytical techniques.

    30. Question 30. What Is Chromatography?

      Answer :

      It is technique for rapid and efficient separation of components of a mixture and purification of compounds. It is based on differential migration of the various components of a mixture through a stationary phase under the influence of a moving phase.

    31. Question 31. What Is The Basis (principle) Of Chromatographic Process?

      Answer :

      It is based on the differential migration of the individual components of a mixture through a — stationary phase under the influence of a moving phase.

    32. Question 32. What Type Of Solvents Are Generally Employed In Chromatography?

      Answer :

      Generally solvents having low viscosities are employed in chromatography. This is due to the fact that the rate of flow of a solvent varies inversely as its viscosity.

    33. Question 33. Name Some Chromatographic Techniques?

      Answer :

      Paper chromatography, column chromatography, thin layer chromatography, gas chromatography. 

    34. Question 34. What Are The Moving And Stationary Phases In Paper Chromatography?

      Answer :

      Water absorbed on cellulose constituting the paper serves as the stationary phase and organic solvent as moving phase.

    35. Question 35. What Is Meant By The Term Developing In Chromatography?

      Answer :

      During chromatography, if the components to be separated are colourless, then these separated components on chromatogram are not visible. Their presence is detected by development, which involves spraying a suitable reagent (called developing reagent) on the chromatogram, or placing the chromatogram in iodine chamber when various components become visible. This process is called developing of chromatogram.

    36. Question 36. How Does The Liquid Rise Through The Filter Paper?

      Answer :

      By means of capillary action.

    37. Question 37. What Is Meant By The Term Rf Value?

      Answer :

      Rf (retention factor) of a substance is defined as the ratio of the distance moved up by the solute from the point of its application to the distance moved up by the solvent from the same point.

    38. Question 38. On What Factors Does The R Value Of A Compound Depend?

      Answer :

      1. Nature of the compound. 
      2. Nature of the solvent. 
      3. Temperature. 

    39. Question 39. Give The Biochemical Uses Of Chromatography?

      Answer :

      It helps in the separation of amino acids, proteins, peptides, nucleic acids, etc.

    40. Question 40. Name The Scientist Who Introduced Chromatographic Technique?

      Answer :

      Russian botanist M. Tswett (1906). 

    41. Question 41. What Are The Advantages Of Chromatography Over Other Techniques?

      Answer :

      1. It can be used for a mixture containing any number of components. 
      2. Very small quantities of the substances can be effectively detected and separated from a mixture. 

    42. Question 42. What- Is Loading (or Spotting)?

      Answer :

      The application of the mixture as a spot on the original line on the filter paper strip or addition of mixture to the column, is called loading (or spotting).

    43. Question 43. What Are The Essential Characteristics Of The Substance Used As A Developer?

      Answer :

      1. It should be volatile. 
      2. It should impart colour to the different spots. 
      3. It should not react with various compounds which are being separated. 

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