1. WHAT IS THE PRINCIPLE OF CHROMATOGRAPHY (HPLC AND GC)?
Chromatography is a separation technique based on the differential distribution of components between two phases: the stationary phase and the mobile phase.
Components interact differently with these phases and therefore travel at different rates, resulting in separation.
- HPLC: Liquid mobile phase and stationary phase (solid support with an appropriate stationary-phase material).
- GC: Gas mobile phase and stationary phase, which may be a liquid film or a solid adsorbent.
2. WHAT ARE REVERSE-PHASE AND NORMAL-PHASE CHROMATOGRAPHY?
Both are chromatographic techniques distinguished by the polarity of their stationary and mobile phases.
Reverse-Phase Chromatography (RP-HPLC)
- Stationary phase: Non-polar (e.g., C18, C8).
- Mobile phase: Relatively polar (e.g., water, buffer, methanol or acetonitrile).
Normal-Phase Chromatography
- Stationary phase: Polar (e.g., silica).
- Mobile phase: Relatively non-polar (e.g., hexane).
3. ARE CHIRAL CHROMATOGRAPHY AND NORMAL-PHASE CHROMATOGRAPHY THE SAME?
No. They are different chromatographic concepts.
Chiral Chromatography:
Used to separate and quantify enantiomers using a chiral stationary phase or a suitable chiral selector.
It can be performed under normal-phase, reverse-phase or other suitable chromatographic conditions.
Normal-Phase Chromatography:
Uses a polar stationary phase and a relatively non-polar mobile phase.
It can be used for the separation of different compounds, including isomers, based on their interactions with the stationary and mobile phases.
Key Difference: Chiral chromatography refers to separation based on stereochemical differences, whereas normal-phase chromatography refers to the polarity arrangement of the chromatographic phases.
4. WHAT IS THE DIFFERENCE BETWEEN CHROMATOGRAPHIC PURITY AND RELATED SUBSTANCES BY HPLC?
Chromatographic Purity:
Chromatographic purity generally refers to the proportion of the main component relative to the total detected chromatographic components, commonly evaluated using area normalization where appropriate.
The result depends on the method’s detection capability and response factors.
Related Substances:
Related substances testing is used to detect and quantify specified and unspecified impurities in a pharmaceutical substance.
It may include both known and unknown impurities.
Quantification may be performed using impurity reference standards, relative response factors or other procedures specified in the applicable monograph or validated method.
Key Difference: Chromatographic purity is a general assessment of chromatographic composition, whereas related substances testing is a defined impurity-control procedure with specified reporting and acceptance criteria.
5. WHAT ARE THE DIFFERENT IMPURITY QUANTITATION METHODS IN HPLC?
The commonly used methods are:
- Area normalization method.
- Internal standard method.
- External standard method.
- Standard addition method.
The selection depends on the analytical method, impurity characteristics, availability of reference standards and required accuracy.
6. WHAT IS THE DIFFERENCE BETWEEN HPLC PURITY, ASSAY AND POTENCY?
Purity:
Indicates the proportion of the desired substance relative to other components present in the sample. Chromatographic purity may be estimated using area normalization, but it does not necessarily represent absolute chemical purity.
Assay:
Determines the quantity or content of the active substance in the sample using a specified analytical method. Results may be expressed as percentage content, depending on the calculation basis.
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.
Represents the strength or biological activity of a substance relative to an established reference standard. For substances where biological activity is relevant, potency may be determined using biological or other appropriate validated methods.
Potency is not necessarily calculated by subtracting impurities from 100%.
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.
Important: Assay, purity and potency must be interpreted according to their respective analytical methods and reporting bases.
7. WHAT IS A BUFFER SOLUTION? EXPLAIN ACIDIC AND BASIC BUFFERS.
Buffer Solution:
A buffer solution resists significant changes in pH when small quantities of acid or base are added or when the solution is diluted.
Acidic Buffer:
A mixture of a weak acid and its conjugate base, usually supplied as a salt, forms an acidic buffer.
Example:
- Acetic acid (CH₃COOH).
- Sodium acetate (CH₃COONa).
Basic Buffer:
A mixture of a weak base and its conjugate acid, usually supplied as a salt, forms a basic buffer.
Example:
- Ammonia (NH₃).
- Ammonium chloride (NH₄Cl).
8. WHAT ARE PROTIC AND APROTIC SOLVENTS?
Protic Solvents:
Solvents capable of donating a proton or hydrogen-bond donor hydrogen.
Examples:
- Water.
- Methanol.
- Ethanol.
- Acetic acid.
Aprotic Solvents:
Solvents that cannot donate a proton under normal solvent behaviour.
Examples:
- Acetone.
- Acetonitrile.
- Dimethyl sulfoxide (DMSO).
- Dimethylformamide (DMF).
9. WHAT IS THE DIFFERENCE BETWEEN HPLC AND GC?
Both HPLC and GC are chromatographic techniques used to separate and quantify components present in a mixture.
Based on Application
HPLC:
- Suitable for non-volatile and thermally unstable compounds.
- Commonly used for pharmaceutical APIs, impurities and related substances.
- Suitable for a wide range of polar and non-polar compounds.
GC:
- Suitable for volatile and thermally stable compounds.
- Commonly used for residual solvent analysis and volatile impurities.
Based on Instrumentation
| Parameter | HPLC | GC |
|---|---|---|
| Mobile phase | Liquid | Gas |
| Stationary phase | Solid support with stationary-phase material | Liquid film or solid adsorbent |
| Separation mechanism | Adsorption, partition and other interactions | Partition or adsorption |
| Pumping system | High-pressure liquid pump | Carrier gas supply and flow control |
| Column temperature | May be controlled or programmed | Oven temperature can be programmed |
| Common detectors | UV, PDA, RI, FLD, MS | FID, ECD, TCD, NPD, MS |
| Typical application | Non-volatile or thermally sensitive compounds | Volatile and thermally stable compounds |
Important Difference: HPLC uses a liquid mobile phase delivered under pressure, whereas GC uses a gaseous mobile phase controlled through a carrier-gas system.
10. WHAT IS PHASE COLLAPSE? CAN WE WASH A REVERSE-PHASE C18 COLUMN WITH 100% WATER?
Phase Collapse
In certain reverse-phase columns, particularly conventional alkyl-bonded silica columns, exposure to highly aqueous mobile phases can cause dewetting of the stationary phase. This phenomenon is commonly referred to as phase collapse.
In this condition, the aqueous mobile phase does not adequately wet the hydrophobic bonded stationary-phase surface, resulting in reduced retention and changes in chromatographic performance.
Can We Wash a C18 Column With 100% Water?
It depends on the column chemistry.
Conventional C18 columns may undergo dewetting when exposed to 100% aqueous mobile phases. However, specially designed aqueous-compatible C18 columns can operate with highly aqueous or even 100% aqueous mobile phases.
Important: Always follow the column manufacturer’s recommendations. Do not assume that every C18 column can or cannot tolerate 100% water.
11. WHAT IS NEEDLE WASH IN HPLC? WHAT IS THE BEST NEEDLE-WASH COMPOSITION?
Needle wash is used to clean the injector needle to minimize sample carryover between injections. Depending on the autosampler design, needle washing may clean the external surface, internal surface or both.
Needle-Wash Composition
The composition should be selected based on:
- Sample solubility.
- Nature of the analyte.
- Mobile-phase composition.
- Potential carryover.
- Compatibility with the instrument and analytical method.
A suitable organic solvent or aqueous-organic mixture may be used. For difficult-to-remove samples, a stronger wash solvent may be required.
Important: There is no universal needle-wash composition. The wash solvent should be selected and verified through carryover studies.
12. WHAT IS SEAL WASH IN HPLC? WHAT IS THE BEST SEAL-WASH COMPOSITION?
Principle
HPLC pumps contain pistons and seals that prevent mobile-phase leakage from the pump head. During operation, small quantities of mobile phase may pass behind the piston seal. If the mobile phase contains buffers or salts, these may accumulate and crystallize behind the seal. This can cause seal deterioration and reduce pump performance.
Purpose of Seal Wash
Seal wash flushes the area behind the piston seal to remove accumulated salts and solvent residues, thereby extending seal life.
Composition
A commonly used seal-wash solution is a mixture of water and a suitable organic solvent.
For example:
- 90% water.
- 10% methanol or another compatible organic solvent.
The exact composition depends on the instrument manufacturer’s recommendations and the mobile-phase composition.
Important: The 90:10 composition is not a universal requirement. Use a compatible seal-wash solution as specified by the manufacturer.
13. WHY DO PEAKS ELUTE EARLIER WHEN WE INCREASE THE ACETONITRILE PERCENTAGE IN REVERSE-PHASE CHROMATOGRAPHY?
In reverse-phase chromatography, increasing the organic solvent percentage generally increases the elution strength of the mobile phase.
Acetonitrile is a stronger eluting solvent than water in typical reverse-phase conditions.
When the percentage of acetonitrile increases:
- The mobile phase becomes stronger.
- The interaction of analytes with the hydrophobic stationary phase becomes relatively less favourable.
- Analyte retention decreases.
- Peaks elute earlier.
Important: In reverse-phase chromatography, increasing the organic solvent percentage generally decreases retention time.
The same principle does not apply directly to normal-phase chromatography, where increasing the polarity of the mobile phase generally increases elution strength.
14. WHAT ARE THE DIFFERENT DETECTORS USED IN GAS CHROMATOGRAPHY? NAME AT LEAST FIVE.
Commonly used GC detectors include:
- Flame Ionization Detector (FID).
- Electron Capture Detector (ECD).
- Thermal Conductivity Detector (TCD).
- Photoionization Detector (PID).
- Flame Photometric Detector (FPD).
- Nitrogen-Phosphorus Detector (NPD).
- Mass Spectrometry Detector (MS).
The selection depends on the nature of the analyte and the required sensitivity and selectivity.
15. WHAT ARE THE ICH Q3C LIMITS FOR THE FOLLOWING RESIDUAL SOLVENTS?
The following are commonly cited Option 1 concentration limits for Class 2 residual solvents under ICH Q3C, expressed in ppm and percentage w/w.
| Solvent | ICH Class | Limit (ppm) | Limit (% w/w) |
|---|---|---|---|
| 1,4-Dioxane | Class 2 | 380 | 0.038% |
| Cyclohexane | Class 2 | 3,880 | 0.388% |
| Pyridine | Class 2 | 200 | 0.020% |
| Xylene | Class 2 | 2,170 | 0.217% |
Important: These are Option 1 concentration limits, based on the assumption of a maximum daily product intake of 10 g. If the daily intake exceeds 10 g, the concentration limits must be evaluated using the applicable permitted daily exposure (PDE) and actual maximum daily dose.
The applicable ICH Q3C version and regulatory requirements should be checked before establishing specifications.