Interview questions in Analytical Chemistry - Part 1
What is FTIR ?
This is the process by which the prepared sample is placed into a mineral oil mull or a Potassium bromide pellet and subjected to infrared radiation light from the infrared part of the light spectrum.
Then infra red light is then partially absorbed/transmitted by the bonding of the molecules within the sample by their stretching and bending.
This absorption is then represented as a spectrum from 450-4000nm.
This is a fingerprint of the substance.
With pure samples clean and best spectrum can be obtained.
At what nanometer noise test for HPLC is performed and why ?
noise test performed at 254 nm, because most of the chromophores absorb and give response at this nanometer.
Tell me the uses of ion pairing agents ?
These are the chemical substances which form complexes with the drug molecules, and for stabilization of the molecule there by aiding in retention to the column. these enhance peak shape and retention time.
The ion pairing agent must be oppositely charged than the analyte and must have good hydrophobicity. Ion-pairing chromatography (IPC) can be used for both positively and negatively charged analytes.
Tell me how can you develop a method for high matrixed samples ?
The development depends on the type of matrix, some time electrophoretic methods are used for the separation of proteins and is time consuming.
Column and fractional chromatography are also good ideas for some molecules.
For some samples GC MS can be employed.
What is the difference between working and reference standard ?
Refrence standads are those which often get from agencies like USP, EDQM, and have COA with the data like NMR and XRPD .
when ever there is large requirement or the minimal availability of compendial standards, working standard can be utilized for the preparation of reference standard by procuring the API raw material from the ware house and check the assay, water content or either LOD and related substances against the reference standard, often do a minimal of duplicate analysis and get the mean there by final potency value and other is determined. Validity or retest usually be one year.
How do we keep the concentration for a sample for RS ?
Initially find the similar wavelength where API and impurities have common response.
If all the impurities have the different response consider the impurity with low response.
or
All the impurities at spec level are having the S/N ratio of more than 30 and then no column overload to be observed.
What are the advantages of Acetonitrile when compared to methanol ?
Acetonitrile is more polar when compared to methanol and provides good resolutions in some cases, and it provides more selectivity.
What are the difference between C8 and C18 columns ?
C18 column has dense packing when compared to C8 column, that leads to more mobile phase travel through the column per unit length.
C8 columns provide short retention times. C18 column retains more than C8 as the interaction of stationary phase with mobile phase is more in C18 providing better seperation.
in general C8 columns provide sharper peaks than C18 columns.
What is the Principle of Karl fischer Volumetric titration ?
In the volumetric titration method,iodine required for reaction with water is previously dissolved in water determination, and water content is determined by measuring the amount of iodine consumed as a result of reaction with water in a sample.
Why Disodium tartarate used for KF Volumetric titrations ?
under normal conditions DST is non hygroscopic and stable having stoichiometric water content of 15.66% so primarily used for titration.
Why methanol used in KF Titrations ?
According to the principle the medium should contain lower organic solvent and need to have the sample dissolving capability and To satisfy sulphur dioxide and Methanol
reaction and formation of methyl sulphite takes place.
Describe the types of detectors used in GC
FID ------- Used for Most organic compounds
TCD ------- Used for Universal detection
ECD ------ For nitrites, halides, chlorides, anhydrites and organometallics.
Nitrogen phosphorus --- For nitrogen and phosphorus detection
Flame photometric --- for sulphur, tin, boron, arsenic, germanium, selenium, chromium.
Photo ionzation detector ------ amines, organo sulphurs, aliphatics, keytones, aromatics and
organo metallics.
When is direct type of injections done in GC ?
These type are done when the sample is in trace amounts and there will be no direct contact with glass wool as the compound is in in trace it can loss through adsorption or degradation.
What are Programmable temperature vapourization injections in GC ?
Here the sample is injected in to a cold inlet and ten it is programmed to increase in temperature often vaporize the solvent to vent and then increasing in temperature to vaporize the compound of interest and introduce them to analytical column.
How do u start the HPLC Method development ?
Every HPLC method development starts primarily with the literature survey available with the molecule.
then we need to find the molecular functionality, polarity and structure from these we can tell whether it comes in RPHPLC or Normal Phase HPLC.
the next step usually would be finding out the lambda max and solubility in various solvents which aid us in deciding the diluent and mobile phase selection.
incase if there we didnot get lambda max we can go for other techniques like derivatization.
Most of the molecules we are using are polar to mid polar hence we will go for RPHPLC.
Initial choice of selection for mobile phase would be Water methanol or water ACN in the ratio of 50:50, this would be feasible for the single impurity API.
Select the columns of general usage like Hypersil columns, Zorbax, Ace, 150 X 4.6mm,5µ columns of C18 type.
start a optimum parameters like 1 ml/min and lambda max obtained from the previous studies would be better and making column temperatures to 25°C.
If there are more impurities available with the molecule we will go for the Gradient type of Elution,
here the parameters like increase in column temperature and decreasing the particle size helps in good resolution of the impurities. increase in the length of the columns often make the run time more.
Coming to buffers, most commonly used ones are phosphate and acetate because of their advantage to be used below 220nm as well and phosphate will have 3 pka values which help in most adjustment over wide ranges.
selecting the pka value of +or- 1 would be optimum for elution's as it provides adequate buffering.
while developing methods view the general parameters like Tailing, resolution and plate count.
Finally after preliminary optimization perform the forced degradation study on the sample as it would illustrate the impurites that will form with excess degradation or in future stability studies.
Describe the zones according to ICH ?
Zone ---- 1 ------- Temperate
Zone -----2-------- Mediterranean/subtropical zone
Zone -----3--------Hot dry zone
Zone------4-------Hot humid/tropical zone
Describe about RRF ?
The pharmaceutical drugs usually contains impurities in them, they might haves same or different response when compared with the main compound. so in order to determine the impurity in the drug it is required to compare with the impurity standard. in some cases impurity standard may not be available so RRF need to be established.
According to ICH Q3 if RRF established correctly it could be used for determining the impurity.
RRF (Relative response factor) is calculated from the response factor of impurity to that of the main compound at a specific nanometer.
Response factor = Peak area
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Concentration in mg/mL
Relative response factor = Response of impurity
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Response of API
Solutions of impurity and standards should be prepared at different concentrations to obtain a plot for its calculation.
What solvents used for the determination of Aldehydes and keytones water content ?
conventionally used solvents form acetals and ketals respectively, resulting in erroneoushigh water content of a molecule.
Reagents like Hydranal-Composite 5 K, Hydranal-KetoSolver used for these types
For coulometric reactions Hydranal-Coulomat AK, Hydranal-Coulomat CG-K