Question 1. What Is Room Temperature?

Answer :25 degree centigrade

Question 2. What Is The Ultraviolet(uv) And Visible Spectroscopy Range?

Answer :UV spectroscopy range 200-400 nm, Visible spectroscopy range 400 nm to 800nm.

Question 3. What Is The Use Of Uv Spectroscopy?

Answer :Spectroscopy used for detecting the functional groups, impurities. Qualitative and
quantitative analysis can be done.

Question 4. What Is The Difference Between Qualitative And Quantitative Analysis?

Answer :Qualitative analysis involves identification of the compound or chemical based on

their chemical(absorption, emission )or physical properties(e.g Melting point, boiling point).

Quantitative analysis involves estimation or determination of concentration or amount of

the chemical compounds or components.

Question 5. Explain The Principle Of Ultraviolet Spectroscopy?

Answer :UV spectroscopy uses light in the UV part of electromagnetic spectrum. UV

absorption spectra arises in which molecule or atoms outer electrons absorb energy,
undergoes transition from lower energy level to higher energy level. For each molecule,
absorbance at wavelength is specific.

Question 6. Explain About Beer Lamberts Law?

Answer :It states that the intensity of monochromatic light absorbed by a substance
dissolved in a fully transmitting solvent is directly proportional to the substance
concentration and the path length of the light through the solution.
Question 7. Explain The Infrared Spectroscopy Principle?
Answer :When a molecule absorbs the Infrared radiation, it vibrates and gives rise to
packed Infrared(IR) absorption spectrum. This IR spectrum is specific for every different
molecule absorbing the IR radiation, useful for its identification.
Question 8. What Is The Body Temperature?
Answer :37oCelsius or 98.6oF
Question 9. Define Ph? What Is The Ph Of Blood?
Answer :pH -Negative logarithm of hydrogen ion concentration. Blood pH-7.35 to 7.45.
Question 10. Expand Lcms, Hplc,uplc, Tlc And Gc?
Answer :
o LCMS- Liquid Chromatography
o HPLC- High Performance Liquid Chromatography,
 o UPLC- Ultra High Performance Liquid Chromatography,
o TLC- Thin Layer Chomatography,
o GC- Gas Chromatography.

Question 11. What Is The Hplc Principle?

Answer :It is a technique used for separating the mixture of components into individual
components based on adsorption, partition, ion exchange and size exclusion principles.
Stationary phase and mobile phase used in it. HPLC used for identification, quantification
and purification of components form a mixture.
Question 12. Explain Hplc Instrumentation?
Answer :It involves solvent system, pump, Sample injector, HPLC columns, Detectors and
Recorder. Firstly, solvent(mobile phase) is degassed for eliminating the bubbles. It is
passed through the pump with a uniform pressure. The liquid sample is injected into the
mobile phase flow stream. It passes through the stationary phase identified by the
detectors and recorded.
Question 13. In Reverse Phase Hplc, Which Type Of Stationary Phase Is Used And Give
Example?
Answer :Non polar stationary phase used
Ex: Silica gel C-18
Question 14. What Are The Detectors Used In Hplc?
Answer :UV detector, IR detector, Fluorescence detector, Mass spectroscopy, LC MS etc.
Question 15. How To Calculate Retention Factor In Paper Chromatography?
Answer :Rf = Distance travelled by solute/ Distance travelled by solvent.
Question 16. Define Molarity?
Answer :Number of moles of solute per litre solution. Denoted with “M”
Question 17. Define Molality?
Answer :Number of moles of solute per kilogram solvent. Denoted with “m”
Question 18. Define Normality?
Answer :Number of Number of moles equivalent per litre solution.
Question 19. Molecular Weight Of Oxygen?
Answer :
16
Question 20. Difference Between Humidity And Relative Humidity?
Answer :
o Humidity – Measure of amount of water vapour present in the atmosphere.
o Relative humidity- Water vapour amount exists in air expressed as a
percentage of the amount needed for saturation at the same temperature.
Question 1. What Does A Quality Control Chemist Do?
Answer :
Quality control: testing samples of raw materials or products either in mid-stage or close to
final stage to make sure that they are of high enough quality, teaching the standards and
qualifications, before the product can be mass produced.
o Research and development of products.
o Perform various chemical methods and procedures in the lab.

Question 2. Specifically, What Company Do You Work For, What Is Your Official
Title/position, And What Are Your Duties?
Answer :
Example:
Lead chemist: works in the lab, chemical reactions, testing the products, analyzing,
quality control of raw materials to match all the qualifications/standards, quality control
of final product, whereas the chemical engineer does the processing and development of
the product.
Company: Javo-Mex (soap/detergent/body wash/hand soap/cleaning products company;
manufacture for P&G, Mr. Clean)
Question 3. Could You Describe Your Typical Day At Work?
Answer :
o Take samples of raw materials or the finished products to the lab for analysis.
o Follow different chemical methods to do analysis to make sure samples have
reached qualifications.
o If not reached qualifications, must tell the supervisors and stop production to
find the problem.
o Inspect other co-workers.
o Fill out a sample report (raw materials, final products have different reports).
o If the raw material/final product/mid-stage product is poor: give the report to
another department.
o Once final product is approved, send to packaging department and then they
will ship it out.
o Once raw materials are approved, send to processing/manufacturing
department to make the product.
Question 4. What Is Your Work Environment Like?
Answer :
o In a company laboratory.
o Must always be sanitary.
o Sometimes if there were a problem, had to go to manufacturing department.
o Go to warehouse to sample the raw material.
o Get finished products from the production/manufacture line.
 Question 5. Who Do You Work With Directly?
Answer :
o Director of the quality control department.
o Chemists, chemical engineers, mechanical engineers, electrical engineers (if
manufacturing has problems, they may consult me, or chemist consults them).
Question 6. What Are Some Of The Challenges You Face At Work And How Do You
Overcome Them?
Answer :Understanding all the chemistry, following the manufacturing/production rules
and standards.
When something is wrong with the raw materials:
If sample is not good, call the raw material manufacturing company, and send back the
materials.
When something is wrong with the final products:
o If sample is not good, keep hold of product and find out the problem by
contacting the different departments.
o Solving the problem is hard; meeting with different departments to solve the
problem.
o Tracing back to the processing stages of product.

Question 7. Around How Many Hours Do You Work Per Day, And How Many Days Per
Week?
Answer :
o During the "high season" (summertime; higher demand) : overtime --> 10 hours
or more
o Regular season: 8 hrs
o During "slow season”: 8 hrs
Question 8. What Is Your Favourite Part About Being A Chemical Engineer?
Answer :
o The chemistry knowledge you get to apply
o The technology
Question 9. What Is Your Least Favourite Part About Being A Chemical Engineer?
Answer :
o Exposure to toxic/poisonous materials (not healthy for the workers).
o Boring routine, doing same thing over and over, monotonous.
Question 10. What Are Your Skills And Attributes That Make You Fit For This Job?
Answer :
o Knowledge of chemistry
o Chemistry or chemical engineering degree
o Communication
o Attention to detail
Question 11. Can You Go Over The Qc Chemist-specific Technology You Use?
Answer :Different types of instruments: HPLC (high performance liquid chromatography),
Infrared spectrum, viscometer (specifically rotational), colorimeter, pH meter.
Use computer analysis with HPLC.
Question 12. What Are Some Of The "chemicals And Substances" That You Utilize And What
Are They Used For?
Answer :
sodium hydroxide --> adjust pH, hydrochloric acid --> adjust pH, sodium chloride -->
adjust the viscosity (for hand soap), different kinds of pigments to adjust fragrance and
colour, various strong acids and bases, propylene glycol and sodium xylene sulfonate to
adjust viscosity, indicators.
Question 13. Could You Give Me An Example Of The Methods You Use In Quality Control?
Answer :
o For detergent: check the colour, smell the odour.
o Check the pH is in the range using pH meter: specific ranges for specific
products.
o Specific gravity is in the range.
o Check the viscosity with viscometer.
o Wet chemical method/titration used to check percentage of efficient chemical
and concentrations.
o Micro confirmation test: Make sure no bacteria in the product.

Question 14. Can Inform Me About Your Educational Background And Credentials, Such
As What Undergraduate Degree You Graduated With, Master's Degree, Professional
Degrees, Etc.?
Answer :
o Master’s degree in chemistry (specifically in polymer) at University of Detroit.
o Undergrad degree in organic chemistry in China.
Question 15. Which Chemistry Courses Did You Find The Most Important For Your
Career?
Answer :
o Organic/inorganic chemistry
o Analytical chemistry
o Physical chemistry
Question 16. What Kind Of A Chemistry Foundation Do You Need For This Career?
Answer :
Good understanding of organic chemistry, and inorganic chemistry, math/calculations.
Question 17. What Specific Topics In Chemistry Does A Qc Chemist Need A Good Grasp
On?
Answer :
Chemical news, mostly inorganic and organic chemistry.
Question 18. What Kind Of Training Is Needed Before Starting The Job?
Answer :
o The company trains me.
o Specific to the company; must follow the company's methods and tests and their
qualifications/standards.
o Lab skills and certain methods were required before one could take the job.
o Familiarize with safety procedures and health hazards during the job.
o Proper lab technique was perfected with more practice, and consistently doing
job.
Question 19. What Are Some Of The Health Hazards In Your Workplace, And What Are
The Safety Procedures You Need To Know?
Answer :
o Wear goggles, do reactions in the fume hood, wear gloves, lab coat, safely
dispose of chemicals (biohazard us waste).
o Benzene, sulphur iC acid had to be handled with care.
o Material safety data sheets had to read.
Question 20. What Do You Think Is The Future For Quality Control Chemist?
Answer :
o Job demand will be strong; because many manufacturing companies need them
to "make sure everything is correct".
o Demand for high quality/safe products always high.
o Or else products will not be the right quality and potentially dangerous to the
public.
o High standards for cleaning/hygiene products calls for more QC chemists.
Question 21. How Do You Think Your Field Will Advance As Technology Advances?
Answer :
o Using more highly technological instruments rather than traditional lab
techniques.
o More efficient, more accurate, faster.
o Have to learn more new technology (computers, instruments).
o Will become large part of career.

Question 1. What Are The Main Differences Between High Performance Liquid
Chromatography And Gas Chromatography?
Answer :
o In HPLC the mobile phase is a liquid whereas in Gas Chromatography the
mobile phase or carrier is a gas.
o 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.
o Gas Chromatography is applied for analysis of volatile compounds whereas non
volatile compounds can be easily analyzed on HPLC
 o 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
o HPLC requires higher operating pressures than GC because liquids require
higher pressures than gases for transport through the system
o HPLC columns are short and wide in comparison 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 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.
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.
Question 4. What Are The Desirable Characteristics Of A Gc Detector ?
Answer :
The detector chosen for particular analysis should :
o Give reproducible response to changes in concentration of eluting compounds
in the carrier gas stream.
o Should provide a large linear dynamic range
o Should have high sensitivity
o Should have small internal volume to give narrow peaks and also facilitate
flushing of previous sample traces
o Should preferably be non-destructive

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

Answer :
Detectors falls into three categories depending upon response to the eluting compounds:
o Non-selective – Respond to all component in the gas stream except for the
carrier gas
o Selective – Respond to a particular class of compounds with common physical
or chemical properties
 o Specific – Respond to a single specific compound only in the carrier gas stream

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
o Wall coated open tubular (WCOT) – Internal wall is coated with a very fine
film of adsorbing liquid
o 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
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.
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.
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.
Question 10. What Measures You Would Adopt To Extend Useful Life Of A Column?
Answer :
o Condition a column before first use or after long time storage
o Take care not to exceed upper temperature limit specified by the
manufacturer
o Avoid injection of solutions which are strongly acidic or basic in nature
o Rinse columns by injection with blank solvents such as methanol, methylene
chloride or hexane to remove contamination of column after excessive usage

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.
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.
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.
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.
o A common non-specific method is suspension of developed chromatogram in
iodine vapour. Most organic compounds show up as brown spots.
o 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.
o 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.
 o Alkaloids – Dragendroff’s reagent spray results in orange or orange yellow
spots.
o Aldehydes&Ketones – 2,4-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 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.
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.
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.
o Ascending paper chromatography – the carrier liquid moves from bottom
upwards.
o Descending paper chromatography – the carrier liquid trough is on top and
mobile phase moves downward on the filter paper.
o 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.
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:
o Solvents are not toxic or carcinogenic.
 o Solvent constituents of mixture should not react with any of the sample
constituents.
o Solvents selected should not interfere in detection of separated spots.
o Solvents should not be highly volatile as loss of components can result in
change of mixture composition.
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:
o Low cost of analysis and freedom from maintenance.
o Separated spots are visible for coloured compounds and colourless compounds
can be viewed by using alternate techniques.
o Minimum operation and training requirements.Solvent consumption is much
less as compared to more sophisticated techniques.
o Paper chromatography serves as a good demonstration of basic concepts of
separation for school and undergraduate students.
Question 20. What Are The Limitations Of Paper Chromatography Technique?
Answer :
Paper chromatography has some limitations such as:
o Semi-quantitative in nature.
o Overlapping of spots of components having close Rf values.
o Higher concentration of components often leads to streaking instead of well-
defined spots.
o 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.
o 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.
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.
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.
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.
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.
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.
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.
Question 27. What Are The Desirable Features Of A High Performance Liquid
Chromatography Detector?
Answer :
The desirable features of a detector are
o Sensitivity towards solute over mobile phase.
o Low dead volume to eliminate memory effects
o Low noise
 o Low detection limits
o Large dynamic linear range
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.
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.
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.
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.
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.
Question 33. Name Some Chromatographic Techniques?
Answer :
Paper chromatography, column chromatography, thin layer chromatography, gas
chromatography.
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.
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.
Question 36. How Does The Liquid Rise Through The Filter Paper?
Answer :
By means of capillary action.
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.
Question 38. On What Factors Does The R Value Of A Compound Depend?
Answer :
o Nature of the compound.
o Nature of the solvent.
o Temperature.
Question 39. Give The Biochemical Uses Of Chromatography?
Answer :
It helps in the separation of amino acids, proteins, peptides, nucleic acids, etc.
Question 40. Name The Scientist Who Introduced Chromatographic Technique?
Answer :
Russian botanist M. Tswett (1906).
Question 41. What Are The Advantages Of Chromatography Over Other Techniques?
Answer :
o It can be used for a mixture containing any number of components.
o Very small quantities of the substances can be effectively detected and
separated from a mixture.
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).
Question 43. What Are The Essential Characteristics Of The Substance Used As A
Developer?
Answer :
o It should be volatile.
o It should impart colour to the different spots.
o It should not react with various compounds which are being separated.
Question.1 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.

Question.2. 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.

Question.3. 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.

Question.4. Name some chromatographic techniques.

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

Question.5. 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.

Question.6. 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.

Question.7. How does the liquid rise through the filter paper ?
Answer. By means of capillary action.

Question.8. 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.
Question.9. On what factors does the Rf value of a compound depend ?
Answer.

Nature of the compound.

Nature of the solvent

.Temperature.

Question.10. Give the biochemical uses of chromatography.

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

Question.11. Name the scientist who introduced chromatographic technique.

Answer. Russian botanist M. Tswett (1906).

Question.12. 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.

Question.13. 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).

Question.14. What are the essential characteristics of the substance used as a developer ?
Answer.

It should be volatile.

It should impart colour to the different spots.

It should not react with various compounds which are being separated.
Question 1 – 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.

Question 2 – 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.

Question 3-. 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

Question 4-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.

1. A common non-specific method is suspension of developed chromatogram in iodine

vapour. Most organic compounds show up as brown spots.
2. 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.
3. 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.
4. Alkaloids – Dragendroff’s reagent spray results in orange or orange yellow spots.
5. Aldehydes&Ketones – 2,4-DNPH spray in methanol and sulphuric acid results in
orange or yellow spots.
Question 5– 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.

Question 6-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.

Question 7 – 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.
 Two – dimensional paper chromatography – After developing the chromatogram in
either ascending or descending mode it is taken out, dried and developed again after
turning by 90° in either the same liquid or another liquid mobile phase.The spots
spread across the sheet and closely overlapping spots get resolved.
 Circular paper chromatography – the mobile phase moves radially outwards from the
centre of a circular piece of paper. In this mode the mixture components get resolved
radially.
Question 8 – 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.

Question 9-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:

o Low cost of analysis and freedom from maintenance.

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

Question 10-What are the limitations of paper chromatography technique?

Answer – Paper chromatography has some limitations such as:

o Semi-quantitative in nature.
o Overlapping of spots of components having close Rf values.
o Higher concentration of components often leads to streaking instead of well-defined
spots.
o 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.
 o 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.
o Filed Under: Paper Chromatography

Question 1. What Is Hplc?

Answer :
HPLC stands for High-Performance Liquid Chromatography (formerly referred to as High-
Pressure Liquid Chromatography).
It is a chromatographic technique used to separate the components in a mixture, to
identify each component, and to quantify each component.
In general, the method involves a liquid sample being passed over a solid adsorbent
material packed into a column using a flow of liquid solvent. Each analyte in the sample
interacts slightly differently with the adsorbent material, thus retarding the flow of the
analytes. If the interaction is weak, the analytes flow off the column in a short amount of
time, and if the interaction is strong, then the elution time is long.

Question 2. What To Do When Back Pressure Increases?

Answer :
o An increase in back-pressure usually suggests either a guard or analytical
column problem. To find exactly where the problem lies we suggest you remove
the guard column (if you are using one) and replace the old cartridge with a
new one.
o If the original pressure is restored, you solved the problem.
o If the pressure remains high, disconnect the analytical column from the system,
backflush it (do NOT connect the column to the detector while doing so) and run
a few column volumes of your mobile phase through the column.
o If the problem still persists you may have some strongly retained contaminants
in your column coming from your previous injections.
o Run the appropriate restoration procedures, as suggested by the column
manufacturer, and retest the column.
o If the initial pressure is not restored you may have to change the inlet frit or
replace the column.
o Always run your system (2 to 5 ml/min) without the guard column and the
analytical column to verify that your pressure isn’t coming from another source,
like a blocked in-line column prefilter, blocked/kinked tubing, particulates
blocking your injector etc.
o Always work your way from the detector back to the pump to isolate the
problem.
Question 3. How Do I Determine The Void Volume In Hplc?
Answer :
The void volume of a system is usually determined by injecting an unretained standard
(Uracil in RP-HPLC) that has no or very little retention on a particular phase. Slight
variations in this value are explained by the extra column dead volume of your specific
system configuration and set-up.
Multiply the elution time of the unretained compound by the flow rate to get the actual
void volume of the system and column. To determine the column void volume alone you
would need to subtract the system void volume determined without the column attached.
Question 4. Why Should I Use A Guard Column With My Analytical Or Preparative
Column?
Answer :
o A guard column is recommended to protect the analytical/preparative column
from contamination from particulates from the injection, debris from worn
pump seals/injector rotor seals or unfiltered mobile phases.
o Filtration through a 0.22um to 0.45 um should be done in order to remove
particles and help degas the mobile phase at the same time.
o Solid Phase Extraction or Liquid Liquid Extraction also help produce a cleaner
sample for direct injection.
o Failure to use a guard column directly exposes the analytical or preparative
column to contamination and therefore reduces its practical lifetime.
o Care should be taken to use, whenever possible, the same material in the guard
column as in the analytical/preparative column especially when doing method
development.
o Typical analytical guard columns are 1 or 2 cm long with either 2.0 (2.1) or 4.0
(4.6) mm depending on the column dimensions and 1cm long for 10mm,
21.2mm & 30mm column id.
o Whether you opt for 1 or 2 cm long guard columns is tied to how harsh your
mobile phase is and how messy your sample is. Ideally, in order to keep your
chromatography and to avoid increasing the system pressure, you should use
the shortest guard column available and use the same id as the column
whenever available. Otherwise you should choose the closest smaller id guard
column available.
Question 5. What Happens If My Sample Solvent Is Stronger Than My Mobile Phase?
Answer :
We do not recommend injecting in a stronger solvent because it usually results in peak
distortion, broadening, poor sensitivity, and shortening of retention times.
This happens because some analytes will tend to travel too quickly through the column,
instead of eluting in a symmetrical band.
If you absolutely must do this, keep the volume as small as possible and make sure the
solvents are miscible.
Question 1. What Is Ph?
Answer :
PH is a measure of the acidity and alkalinity properties of an aqueous solution, which are
determined by the concentration of hydrogen ions (H+) in the solution. Water has the
molecular formula of H2O and almost always exists in the stable molecular state of H2O.
However, a small proportion also exists in the form of ions known as hydrogen ions (H+)
and hydroxyl ions (OH–), and the balance between these hydrogen ions and hydroxyl ions
determines the pH. The solution becomes acidic if there are many hydrogen ions, while it
is alkaline if there are many hydroxyl ions.pH Analyzer Working Principle
Because the product of hydrogen ions (H+) and hydroxyl ions (OH–) is constant in any
aqueous solution at the same temperature, by measuring the concentration of hydrogen
ions (H+) it is possible to determine the degree of acidity or alkalinity. Generally, pH is
defined as:
pH=-log10［H+］
The hydrogen ion concentration of a neutral solution is mol/L, that of a 1/10 mol/L HCl
solution is mol/L, and that of a 1/10 mol/L NaOH solution is mol/L, which are very small
and inconvenient for practical use. Therefore, the inverse of the hydrogen ion
concentration represented as a common logarithm is used as the pH.
For a neutral solution, the pH is 7 because [H+] = ; for a 1/10 mol/L HCl solution, the pH is
1 because [H+] = ; and for a 1/10 mol/L NaOH solution, the pH is 13 because [H+] =10-13
Question 2. How Is Ph Measured?
Answer :
Several methods of measuring pH have been developed and upgraded. Currently, the
glass electrode method is most often used in various fields.
Indicator method:

Hydrogen electrode method:

Quinhydrone electrode method:
Antimony electrode method:
Glass electrode method:

Question 3. What Is Temperature Compensation Of Ph Meters?

Answer :
The emf generated by a pH sensor changes depending on the temperature of the test
solution. This is because the potential generated on the glass membrane of the pH sensor
is proportional to absolute temperature T as represented by Nernst’s equation.
Temperature compensation is done to compensate these temperature-based changes in
the emf with the pH analyzer (or pH transmitter) so that it is independent of temperature.
Note that this temperature compensation is unrelated to changes in the pH value of a test
solution caused by temperature.
Some pH meters measure temperature with an RTD incorporated in a pH sensor to
automatically conduct temperature compensation of the generated emf of the pH sensor.
Question 4. What Is Temperature Compensation (reference Temperature Conversion) Of
A Test Solution?
Answer :
For general pH measurements, changes in the pH value of a test solution caused by
temperature will not be compensated. However, for deionized water measurement, the
temperature of a test solution may be compensated independently of the temperature
compensation of a pH sensor.
The pH sensor for high-purity water measures the temperature using the RTD
incorporated in the pH sensor and can perform the temperature compensation of the emf
generated by the sensor and the temperature compensation of a test solution
simultaneously.
Question 5. What Is Alkaline Error?
Answer :
In the alkaline region of pH10 or more, the emf of a glass electrode deviates from the
linear value on the alkaline side. This is called the “alkaline error.”
Because the magnitude of the alkaline error varies depending on the glass membrane
compositions, a glass electrode for high alkalinity should be used if the pH sensor is used
in the alkaline region. However, this does not guarantee that the alkaline error is
eliminated.
6. What Is Acid Error?
Answer :
The acid error occurring in the region of pH3 or less depends on the glass membrane
composition and the types of acids. Once a glass electrode has acid error, it cannot be
restored quickly even by immersing it in a neutral solution; recovery takes a
considerable time. However, the acid error is small compared to the alkaline error and so
is unimportant for practical use. Question
Question 7. How Is A Ph Analyzer Calibrated With The Standard Solution?
Answer :
To perform pH measurement, the pH meter must always be calibrated using the standard
solution. The general calibration method is “two-point calibration” which is performed
using two types of pH standard solution, but the simple “one-point calibration” is
conducted using one type of pH standard solution.
As it is also called a “buffer solution,” a pH standard solution has the property (buffer
action) of protecting against the addition of an acid or base and minimizing pH changes.
The types of pH standard solutions and pH values at each temperature are specified in
“Methods of pH Measurement.”
Question 8. What Is Zero Calibration (asymmetry Potential Adjustment
Answer :
The interior of a glass electrode is filled with a pH7 solution. If the glass electrode is
immersed in a pH7 solution, the potential difference should be 0 mV because identical
buffer solutions are present on both sides of the membrane of the glass electrode. In
practice, a potential develops due to the strain caused during glass production, shape,
glass compositions, or other factors. This is called the “asymmetry potential.” The
asymmetry potential also occurs due to contamination of the internal solution of the
reference electrode, dryness of or clogging in the liquid junction, etc. in addition to the
glass electrode. To eliminate this asymmetry potential, a pH sensor is zero calibrated
using the pH7 standard solution (pH standard solution of neutral phosphate).
Question 9. What Is Span Adjustment (electric Potential Gradient Adjustment)?
Answer :
Actually, the emf per pH of a glass electrode is not always equal to the value of
2.3026RT/F in the Nernst equation. Therefore, a pH meter requires compensation for
small differences from the theoretical slope of potential, which can be done by
adjustment using a pH 4 or pH 9 standard solutions. This is called span adjustment.
Question 10. How Should Ph Standard Solutions Be Stored, And For How Long?
Answer :
Prepared standard solutions should be stored in hermetically sealed, high-quality hard
glass or polyethylene containers. The pH value of the standard solution may
change(Note) after long-term storage. Before using a standard solution that has been
stored for a long time after preparation, compare its pH with that of a freshly prepared
standard solution and ensure that they are the same.
Moreover, a pH standard solution must never be used once it has been left to the
atmosphere.
Note: For example, borate and carbonate pH standard solutions may absorb CO2 and
other substances in the atmosphere and their pH values may decrease.
Question 11. What Are The Conditions Of A Test Solution?
Answer :
The ranges of test solution temperature and test solution pressure vary depending on the
types of pH sensor, holder type, and holder material.
Question 12. What Are The Orp Analyzer And What Is It Used For?
Answer :
ORP (Oxidation Reduction Potential) refers to the potential that arises due to electron
movement at the time of oxidation and reduction. It is also called Redox (Reduction
Oxidation Potential). In this process, a variety of chemical reactions are used for
refinement, removal, separation and the like, and oxidation reduction is one of these
reactions. The ORP value is an effective index for process management such as detecting
the end of a reaction. The major applications include water discharge for plating, removal
of chlorine gas, sewage treatment and the like.
Question 13. How Can I Get Reliable Ph Measurements?
Answer :
Here are some basic steps that will help you get better, more reliable pH measurements:
o Soak new electrodes before use! A 50/50 mixture of pH 4 buffer and saturated
potassium chloride is an excellent soak solution.
o Reference electrode fruits must be free-flowing to allow internal solution to
make contact with the sample solution. Maintain the fruit in a clean condition.
o Use fresh buffers for calibration.
o Temperature impacts electrode performance. Use temperature compensation,
or keep all samples and standards at the same temperature.
o Calibrate frequently.
Question 14. What Is Automatic Temperature Compensation (atc)?
Answer :
The Solution Temperature Effect:
When there is an increase or decrease in the temperature of a solution, the pH of the
solution can change. This change is not an error caused by the variation in temperature; it
is the true pH of the solution at the new temperature. Since this is not an error, there is no
need to correct or compensate for the solution temperature effect.
The pH Electrode Temperature Effect:
There is only one major temperature effect in pH measurement that can cause errors in
readings. This is the change in the electrode’s response (or sensitivity) to pH which
results from changes in the samples temperature. It is the only reasonably predictable
error due to changes in temperature, and is the only temperature related factor that pH
instruments with temperature compensation can correct for. This temperature error is
very close to 0.003 pH/°C/pH unit away from pH 7. If a sample is measured without using
an automatic temperature probe, the solutions temperature needs to be entered into the
meter manually to allow it to account for this error.
Question 15. How Should An Electrode Be Stored When Not In Use?
Answer :
Electrodes should be stored in electrode storage solution (025 192) between readings
and for short–term storage. If storage solution is not readily available, liquid–filled
electrodes can be stored in pH 4.0 buffer solution. To return an electrode to long term
storage, prepare it in the same condition in which you received it; usually, this means
simply moistening and replacing the end cap of gel–filled electrodes to protect and keep
the sensing membrane active. To store liquid–filled reference and combination
electrodes, refill with electrolyte, cover the fill hole, and moisten and replace the
protective plastic cap.
Important note: Never store an electrode in distilled or deionised water. This may lead to
slow, sluggish response.
Question 16. How Should A Ph Electrode Be Cleaned?
Answer :
A dirty glass membrane is usually indicated by beads of water forming on the bulb when
it’s rinsed with distilled water.
The bulb can be cleaned as follows:
 o For protein layers, soak in a freshly prepared solution of 1% pepsin in 0.1N HCl
for 30 minutes.
o For inorganic deposits, wash with a 1M EDTA solution, 2M ammonia, or 2M acid.
o For grease and similar films, wash with acetone, methanol, etc.
Question 17. How Should An Electrode Be Reconditioned?
Answer :
Prolonged use, excessive alkaline immersion, or high-temperature operation will cause
surface leaching of the membrane glass. The result is extremely noisy and/or sluggish
response, which cannot be remedied simply by cleaning the electrode. If this occurs, the
following procedures will often provide stability and pH sensitivity. Always consider the
electrode’s materials of construction before using these procedures.
o Empty the reference chamber, rinse with deionised water, empty and refill with
the specified filling solution.
o Soak the electrode in hot (50°C – 60°C) reference electrolyte for a few minutes.
o Soak the electrode overnight in pH 4 buffer.
o Remove any exterior salt deposits with distilled water.
o If the filling solution does not flow through the junction by this time (generally
due to an unusually low junction porosity), use gentle suction to pull filling
solution through the junction and repeat from step 2.
o Sometimes the material clogging the junction requires more severe action.
Should the above fail, proceed as follows:
o Use a solvent specific to the solution or material plugging the junction, if
possible.
o Soak the membrane overnight in 0.1 M HCI.
o If measurements have been made in samples containing protein, remove
protein deposits by soaking the electrode bulb in 0.1 M HCl containing 1%
pepsin.
o Repeat from step 1.
If all these fail, the electrode should be discarded safely and replaced.