Interview Questions.

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

Answer :

In HPLC the cellular segment is a liquid while in Gas Chromatography the cellular section or service is a gasoline.
HPLC is useful for analysis of samples which are susceptible to decompose at higher temperatures. GC entails high temperatures so compounds are solid at such temperatures.
Gas Chromatography is carried out for analysis of unstable compounds while non volatile compounds can be without problems analyzed on HPLC
Gas Chromatography can not be used for evaluation of high molecular weight molecules while HPLC has programs for separation and identification of very excessive molecular weight compounds
HPLC requires higher running pressures than GC due to the fact beverages require better pressures than gases for delivery via the machine
HPLC columns are brief and wide in assessment to GC columns
Question 2. Which Type Of Gc Detector Is Most Commonly Used? Explain Its Working Principle And What Are Its Limitations?

Answer :

The maximum normally used detector is the flame ionize detector. The pattern is combusted with the assist of gas gasoline and oxidant within the detector body. Combustible pattern additives burn and bring ions and electrons which can behavior power via the flame. A massive capability difference is carried out at the burner tip and the collector electrode placed above the flame and the modern-day among the electrodes is measured. The detector is mass touchy and reaction isn't tormented by carrier gasoline glide fee modifications. However, the detector isn't always attentive to inorganic gases inclusive of CO, O2, NH3, N2, CS2, CO2, and so on.

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

Answer :

Inert gases commonly used in evaluation when using FID detector are Nitrogen and Helium. Nitrogen is greater normally used as it's far much less highly-priced than Helium. Purity of carrier fuel ought to be greater than ninety nine.995% and online traps need to be used to save you residual moisture or different impurities from coming into the machine.

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

Answer :

The detector selected for unique analysis need to :

Give reproducible response to modifications in awareness of eluting compounds inside the provider gas movement.
Should provide a huge linear dynamic variety 
Should have high sensitivity 
Should have small inner quantity to present slim peaks and also facilitate flushing of previous pattern traces 
Should otherwise be non-damaging
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Question five. What Do You Understand By Specificity Of A Detector?

Answer :

Detectors falls into 3 categories depending upon reaction to the eluting compounds:

Non-selective – Respond to all aspect in the gas flow except for the provider gasoline
Selective – Respond to a particular class of compounds with commonplace bodily or chemical houses 
Specific – Respond to a unmarried precise compound most effective inside the service fuel movement
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Question 6. What Are The Commonly Used Types Of Capillary Columns?

Answer :

Capillary columns are typically 10 – 100m long tubes having an internal diameter ranging from zero.1 – 0.5mm manufactured from bendy fabric along with fused silica. Common varieties of capillary columns are

Wall coated open tubular (WCOT) – Internal wall is covered with a totally fine film of adsorbing liquid 
Surface coated open tubular (SCOT) – Inner wall is covered with a layer of strong aid directly to which the liquid phase is absorbed. 
The columns are bendy and wound into numerous turn coils supported on a SS cage within the column oven

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

Answer :

On non-stop use a column progressively loses its original decision electricity. Column efficiency is expressed on the idea of plate principle idea. Each element below separation spends a finite time in every theoretical plate. Smaller the plate height the bigger the wide variety of plates (N) and better is the column performance.

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

Answer :

Temperature programming means change of temperature of the column at a charge predetermined charge all through the analytical run. This has the equal impact on elution time of separated additives as gradient programming in HPLC analysis. Temperature programming enables reduce analysis time by allowing early elution of much less volatile additives.

Question nine. When Is Isothermal Operation Useful?

Answer :

Isothermal operation is beneficial whilst high decision is required for keeping apart compounds having narrow boiling range. Temperature is ready to round mid variety of boiling factors of ingredients. This outcomes in accurate resolution of low boiling additives however band broadening of better boiling components can result because of their longer retention within the column.

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

Answer :

Condition a column earlier than first use or after long term garage 
Take care not to exceed upper temperature limit designated by way of the manufacturer 
Avoid injection of answers which might be strongly acidic or fundamental in nature 
Rinse columns with the aid of injection with blank solvents such as methanol, methylene chloride or hexane to dispose of contamination of column after excessive utilization
Question eleven. What Is The Basic Principle Of Paper Chromatography?

Answer :

Paper chromatography is a shape of liquid chromatography in which the components of a aggregate of natural compounds get separated as unique spots by way of unidirectional drift of the growing liquid mobile segment solvent mixture over the filter paper to which a spot of the pattern is applied. The distance travelled via each thing is unique beneath the given set of operational conditions.

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

Answer :

The developing liquid section accommodates of a natural solvent however extra frequently it's far a mixture of  or greater solvents in designated proportions. In case solvents are mixed and stored for lengthy periods there can be loss of risky issue so that you can regulate the combination proportions.

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

Answer :

During the chromatogram development chamber is blanketed.This is vital as the environment within the chamber ought to stay saturated with the solvent vapour. Development times can range from approximately an hour to numerous hours and a saturated surroundings prevents losses because of evaporation.

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

Answer :

It is straightforward to differentiate colored spots visually but for colourless compounds alternate strategies need to be followed which can be precise or non-unique.

A common non-unique approach is suspension of advanced chromatogram in iodine vapour. Most organic compounds display up as brown spots.
The sheet is viewed in a UV Viewing cabinet beneath 366 nm and 254 nm wavelength lamp illumination. On commentary the spots need to be carefully marked with a pencil for Rf calculations.
Under precise strategies amines and amino acids are observed by means of spraying heated paper on improvement with zero.2% hydrazine. Deep blue or purple spots begin to appear.
Alkaloids – Dragendroff’s reagent spray outcomes in orange or orange yellow spots.
Aldehydes&Ketones – 2,four-DNPH spray in methanol and sulphuric acid results in orange or yellow spots.
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 must have a medium solubility in the growing solvent aggregate.Too high a solubility will result in transfer of the issue alongwith the solvent the front and alternatively if the solubility is just too low the element will now not be carried by using the solvent aggregate and could continue to be close to the preliminary implemented spot. In either case the decision of the aggregate components will be low. Thus fairly correct resolution can be received for medium solubility of compounds inside the solvent mixture.

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

Answer :

Retardation aspect Rf is a degree of the separation of a specific element. It is expressed as

Rf = distance moved through the thing spot/ distance moved via solvent the front

Rf is a unit less amount and lies between 0and 1.A value of 0 shows no separation has taken area and 1 represents that the component has moved whole length alongwith the solvent the front. In case  spots have same fee of Rf it indicates that they may be no longer resolved. At least a distinction of 0.05 is vital to figure the separation between  spots.

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

Answer :

Paper chromatography separations are labeled according with the direction of go with the flow of mobile section along the filter paper.

Ascending paper chromatography – the service liquid actions from backside upwards.
Descending paper chromatography – the service liquid trough is on pinnacle and cellular segment moves downward on the filter paper.
Ascending – descending paper chromatography – The paper is rolled downward over the rod at the top. On reaching the pinnacle in ascending mode it starts offevolved downward movement within the next section.
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Question 18. What Are Essential Criteria For Selection Of Suitable Solvents For Paper Chromatography?

Answer :

Solvents are selected on the idea of solubility of the pattern components. In trendy it is really helpful to keep in mind:

Solvents aren't poisonous or carcinogenic.
Solvent components of combination have to now not react with any of the pattern elements.
Solvents selected ought to not intrude in detection of separated spots.
Solvents need to not be exceptionally risky as loss of components can result in change of mixture composition.
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Question 19. Why Paper Chromatography Has Retained Its Applicability In The Face Of A Emergence Of Advanced Instrumental Techniques?

Answer :

Chromatographic approach of evaluation has seen an impressive boom over time. Such advances have accelerated laboratory throughputs decreased limits of detection and has made forays into new regions of packages. Paper chromatography has retained its ground until date and is popular in laboratories across the world. Some of the reasons for this are:

Low value of analysis and freedom from maintenance.
Separated spots are visible for colored compounds and colourless compounds can be considered via the use of trade techniques.
Minimum operation and training requirements.Solvent intake is lots less as compared to greater state-of-the-art techniques.
Paper chromatography serves as a very good demonstration of simple principles of separation for school and undergraduate students.
Question 20. What Are The Limitations Of Paper Chromatography Technique?

Answer :

Paper chromatography has a few boundaries along with:

Semi-quantitative in nature.
Overlapping of spots of additives having near Rf values.
Higher awareness of components often leads to streaking in preference to well-described spots.
Errors in Rf calculations can end result from uneven go with the flow of solvent the front. This can be as a result of going for walks out of solvent at the lowest of the chamber, choppy cutting of the filter out paper or unevenness of the bottom of the improvement chamber.
Improper sample spotting, spotting beneath the marked line resulting in dipping into the solvent or unintended dipping of spot into solvent while putting the paper into the solvent chamber.
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Question 21. Which Type Of High Performance Liquid Chromatography Technique Is Most Widely Used?

Answer :

Reverse segment Chromatography has the widest variety of applications. The stationary segment comprises non polar  natural chains certain to inert silica floor and cell section incorporates of aqueous or aqueous-natural combinations  comprising of polar solvents of varying ranges of polarity. The elution collection is polar accompanied through much less polar and least polar or non polar compounds eluting last through the column.

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

Answer :

In length exclusion chromatography the separation does no longer involve chemical interactions between eluting molecules and stationary segment. The separation takes place on the basis of molecular size with larger molecules eluting first and small molecules ultimately. Small molecules are retained longer inside the pores of the desk bound phase therefore they get eluted ultimate.

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

Answer :

Mobile levels entrap air from the ecosystem and this trapped air gets launched as small bubbles underneath high pressures encountered during the HPLC analysis. Such bubbles can lead to noise in detector response or hinder go with the flow of cellular segment via columns. In order to triumph over such problems degassing of cellular phase becomes crucial.

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

Answer :

The maximum normally used detector in HPLC is the UV-VIS detector. The reason for its essential use is that it gives precise response to a selected compound or class of compounds. Most of the natural compounds soak up at unique wavelengths included in the available wavelength variety of the detector.

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

Answer :

A bulk belongings detector responds to a few belongings of cell phase and sample aggregate passing thru it at any point of time such a Refractive index or Electrochemical detector while a particular assets detector is responsive handiest to the feature belongings of the eluting molecule and is impartial of adjustments in cell segment composition together with UV-Vis and Fluorescence detectors.

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

Answer :

When the composition of the cell section isn't always changed via the chromatographic  run  the operation is called as isocratic. It can involve a unmarried solvent or a combination of two or more solvents mixed in a fixed proportion. In gradient operation the composition at begin of run is programmed to change at a predetermined price and the composition on the stop of run is different from the composition on the start.

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

Answer :

The desirable features of a detector are

Sensitivity closer to solute over cellular segment.
Low  useless extent to do away with  reminiscence effects
Low noise
Low detection limits
Large dynamic linear variety
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Question 28. What Do You Understand By Theoretical Plate Concept And How Hetp Affects The Separation Of Hplc Column?

Answer :

Plate theory idea changed into added to provide an explanation for performance of columns. The idea assumes that a nation of on the spot equilibrium exists    among the attention of solute in desk bound segment and the mobile phase and similarly the column is supposed to be divided into some of theoretical plates. Any analyte spends a finite time in every plate and this is the equilibrium time. Smaller the plate peak the greater is the variety of plates in a given duration (HETP) and better is the column resolution.

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

Answer :

Fast or UHPLC approach uses small debris under 2 μ length Use of such particle sizes result in excessive resolution and as small columns may be used  it consequences in of completion of analysis in a whole lot less time thereby lowering consumption of costly solvents.

We wish you had a extremely good learning experience via the introductory unfastened e-getting to know HPLC path. I shall remain in touch with you for our offerings on boost variations of the HPLC e-mastering guides and next introduction masking different analytical techniques.

Question 30. What Is Chromatography?

Answer :

It is technique for speedy and green separation of additives of a mixture and purification of compounds. It is based on differential migration of the diverse additives of a combination through a stationary phase below the impact of a shifting section.

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

Answer :

It is based totally on the differential migration of the man or woman components of a mixture through a — desk bound section below the impact of a shifting section.

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

Answer :

Generally solvents having low viscosities are employed in chromatography. This is because of the truth that the charge of glide of a solvent varies inversely as its viscosity.

Question 33. Name Some Chromatographic Techniques?

Answer :

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

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

Answer :

Water absorbed on cellulose constituting the paper serves because the desk bound section and organic solvent as transferring section.

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

Answer :

During chromatography, if the additives to be separated are colourless, then these separated additives on chromatogram aren't seen. Their presence is detected through development, which entails spraying a suitable reagent (called developing reagent) on the chromatogram, or setting the chromatogram in iodine chamber while various additives turn out to be visible. This method is known as developing of chromatogram.

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

Answer :

By means of capillary action.

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

Answer :

Rf (retention factor) of a substance is described as the ratio of the distance moved up by the solute from the factor of its software to the distance moved up by using the solvent from the identical factor.

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

Answer :

Nature of the compound. 
Nature of the solvent. 
Temperature. 
Question 39. Give The Biochemical Uses Of Chromatography?

Answer :

It facilitates inside the separation of amino acids, proteins, peptides, nucleic acids, and so forth.

Question forty. Name The Scientist Who Introduced Chromatographic Technique?

Answer :

Russian botanist M. Tswett (1906). 

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

Answer :

It can be used for a aggregate containing any range of additives. 
Very small portions of the materials can be efficaciously detected and separated from a aggregate. 
Question 42. What- Is Loading (or Spotting)?

Answer :

The software of the aggregate as a spot on the original line at the filter out paper strip or addition of mixture to the column, is referred to as loading (or recognizing).

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

Answer :

It should be risky. 
It must impart shade to the special spots. 
It have to now not react with various compounds which might be being separated.