QUALITY CONTROL INTERVIEW QUESTIONS ON CHROMATOGRAPHY

       1)WHAT IS THE PRINCIPLE OF CHROMATOGRAPHY (HPLC&GC)?

Separation of, mixture of components, Based on equilibrium distribution of analyte between two immiscible phases (like stationary phase and mobile phase).

HPLC: Solid stationary phase, Liquid mobile phase

GC     : Liquid stationary phase, Gas mobile phase.

       2) WHAT ARE REVERSE PHASE AND NORMAL PHASE CHROMATOGRAPHY?

These are the different type of techniques in chromatography.

Reverse phase chromatography:

Stationary phase: Non-polar

Mobile phase      : Polar

Normal phase chromatography:

Stationary phase: Polar

Mobile phase      : Non-polar

       3)IS CHIRAL CHROMATOGRAPHY & NORMAL PHASE CHROMATOGRAPHY ARE SAME or NOT?

No both are not 100% same.

Chiral chromatography:

             In this we determine the content of chiral isomer whether is it in normal phase or in reverse phase.

Normal phase chromatography:

             In this as above said we must use polar stationary phase and non-polar mobile phase whether is it to determine content of isomer or purity of analyte or whatever it may be.

       4) WHAT IS THE DIFFERENCE BETWEEN CHROMATOGRAPHIC PURITY & RELATED SUBSTANCE BY HPLC?

Chromatographic purity:

              Chromatographic purity is used when all the impurities are known in the given sample. Here we quantitate only known impurities.

Related substance:

               Related substance is used to quantitate both known as well as unknown impurities in the given sample.

       5)         WHAT ARE THE IMPURITY QUANTITATION METHODS IN HPLC?

There are mainly four types.

      1.      Area normalization method

      2.      Internal standard method

      3.      External standard method

      4.      Standard addition method

       6)  WHAT IS THE DIFFERENCE BETWEEN HPLC PURITY, ASSAY AND POTENCY?

Purity:

            It explains how pure our analyte is. It is not related to the amount of analyte present. Simply it is “100-% known or unknown impurity detected in HPLC”

Assay:

           It explains how much of our analyte is (i.e. content of our analyte). It is not related to purity of our analyte.

Potency:

           It explains how potent our analyte is (i.e. the original content of our analyte). It is calculated by mass balance technique. Simply it is 100-all possible impurities.

All possible impurities include Chromatographic impurities (HPLC, GC, and TLC), Heavy metals, Sulphated ash, MC, LOD etc.

Explanation:

            Let us assume an analyte of A with 99.5% purity, 100% assay and 99.0% potency. Then prepare 20%, 50% and 85% solution and analyze in HPLC.

For 20% solution you will get 99.5% purity, 20% assay and 99.0% potency.

For 50% solution you will get 99.5% purity, 50% assay and 99.0% potency.

For 85% solution you will get 99.5% purity, 85% assay and 99.0% potency.

Because we are diluting the concentration so purity will never change for dilution.  Potency is calculated from purity so it is also remains same.

       7)  WHAT IS A BUFFER SOLUTION? CAN YOU EXPLAIN ABOUT ACIDIC BUFFER & BASIC BUFFER?

Buffer solution:

          A solution which can resist change in its pH value on dilution or on the addition of solution of acid or base (Whole pH remains constant) is known as buffer solution.

Acidic buffer:

         Mixture of equimolar quantities of weak acid and salt of this weak acid with a strong base is known as acidic buffer.

Ex: Mixture of CH3COOH & CH3COONa

Basic buffer:

          Mixture of equimolar quantities of weak base and salt of this weak base with a strong acid is known as basic buffer.

Ex: Mixture of NH4OH & NH4Cl

       8)WHAT ARE PROTIC & APROTIC SOLVENTS?

Protic solvents:

              Any molecular solvent which contain dissociable H⁺ ion is called Protic solvent.

(The molecules of such solvents can donate an H⁺  proton.

Aprotic solvents:

              Any molecular solvent which does not contain dissociable H⁺ ion is called Aprotic solvent. (The molecules of such solvents cannot donate an H⁺  proton).

       9) WHAT IS DIFFERENCE BETWEEN HPLC & GC?

Principally both are same. Both are used for the separation of components present in the mixture of sample. Even though these two are principally same, these two also have some differences based applicability, Instrumentation.

Based on applicability:

·         HPLC is used for the separation of thermally stable (or non volatile) compounds.

·         GC is used for separation of volatile (thermally stable) compounds.

In nature 80% of the compounds are non volatile so we use HPLC more frequently than GC.

            Based on instrumentation:

·         In HPLC we use liquid mobile phase and solid stationary phase (Adsorption technique). And in HPLC we use small columns (more frequently up to 30 cm).

·         In GC we use gas mobile phase and liquid stationary phase (Partition technique). And in GC we use columns of length up to 105 cm.

Mobile phase, stationary phase, column, Detector...etc have to select based on sample properties in both the cases.

·         In HPLC we have the pumping system to pump the mobile phase with somewhat high pressure. But in GC we have no pumping system.

·         In HPLC we cannot change the column temperature with time. But we can change temperature with time in GC by giving oven program.

·         In HPLC we use detectors like UV, RI...etc where as in GC we use FID, ECD...etc.

10)  WHAT IS PHASE COLLAPSE? (or)

 CAN I WASH MY HPLC REVERSE PHASE COLUMN WITH 100% WATER?

Before going to this question we should know the C18 column structure. In HPLC C18 column the inner wall of the column is packed with silica (Si-o-Si), this silica is bonded to C18 linkages.

So from this we can clearly understand that our washing solvent will flow on to C18 linkages.

If we wash our Reverse phase C18 column with 100% water, Due to polar (water) and non polar (C18 linkage) repulsion stationary phase matrix collapsed. This collapse is known as PHASE COLLAPSE. So we should not wash our reverse phase column with 100% water.

11)  WHAT IS NEEDLE WASH IN HPLC? (or)

 WHAT IS THE BEST COMPOSITION FOR NEEDLE WASH?

           The needle wash is used to clean the injector needle before and after the injection. The design of needle in the injector system varies for different manufacturers.

In some designs the inside of the needle is part of the flow path of the mobile phase and thus it is flushed continuously by the mobile phase between injections. In this case it is outside (upper layer) of the needle which is cleaned by the needle wash. In some other designs needle is separate from mobile phase flow path and thus the needle wash is used to clean inside and outside of the needle.

         In all the cases composition of needle wash needs to be matched to the sample since this is what you want to clean off the needle. Therefore typically the composition of needle wash is that which matches the proportion of aqueous and organic solvents in the mobile phase will be appropriate.

12)  WHAT IS SEAL WASH IN HPLC? (or)

 WHAT IS THE BEST COMPOSITION FOR SEAL WASH?

Before going to this question first of all we should know about seal in HPLC. In HPLC pump system there is a piston which moves back and forward in the pump head which drawing in and pushing out the mobile phase with each movement. Since it is a moving part, a seal around this piston is required to prevent mobile phase leaking out of the back of the pump, over a period of time small amounts of mobile phase solvents leach through the seal to the back of the piston. If these solvents contain buffers then the salts may precipitate out forming deposits, which can shorten the life of the seal.

So seal wash is to flush the back of the piston seals to remove any deposits and maximizing the lifetime of the seal.

Composition:

It should be aqueous to dissolve buffer and a small amount of organic is required to prevent the bacteria growth and also to reduce the surface tension of water. Typically seal wash composition is 90% water and 10% organic solvent. The organic solvent may be methanol, acetonitrile and IPA…etc.

13)  IN REVERSE PHASE CHRMATOGRAPHY PEAKS ELUTE EARLIER WHEN WE INCRESE ACETONITRILE.WHY?

Most of people confused that, Water is more polar than acetonitrile so when we increase % acetonitrile peaks should elute late because in reverse phase chromatography more polar one comes out first and non-polar comes out last.

In any chromatography the elution order of analyte depends on eluting strength of solvent. In reverse phase chromatography, the less polar, the greater the eluting strength. So the % of ACN increases the eluting strength also increases so the peaks elute earlier. Reverse is true for normal phase chromatography.

Note:

            1.      In above case except %ACN, remaining all same in both the cases.

14)  WHAT ARE DETECTORS USED FOR GAS CHROMATOGRAPHIC ANALYSIS? NAME AT LEAST 5?

     1.      Flame ionization detector.(FID)

     2.      Electron capture detector.(ECD)

     3.      Thermal conductivity detector.(TCD)

     4.      Photo ionization detector.(PID)

     5.      Flame photometric detector.(FPD)

     6.      Nitrogen-Phosphorous detector.(NPD)

15)  GIVE ICH LIMITS OF FOLLOWING MENTIONED SOLVENTS IN % W/W?

      1.      1,4-Dioxane (Class 2) – 380 ppm

      2.      Cyclo hexane (Class 2)  – 3880 ppm

      3.      Pyridine (Class 2) – 200 ppm

      4.      Xylene (Class 2) – 2170 ppm