Analytical Instrument Qualification: A Comprehensive Review on HPLC And UV Systems

Instrument qualification is a systematic procedure that generates documented proof showing that an instrument is suitable for its intended purpose and is properly maintained and calibrated for consistent use. The qualification process is generally divided into four main stages: 

• Design Qualification (DQ) 
• Installation Qualification (IQ)  
• Operational Qualification (OQ) 
• Performance Qualification (PQ) 

Design Qualification (DQ) outlines the user’s requirements and specifies the functional as well as operational features of the instrument. Its purpose is to confirm that the selected instrument possesses the capabilities and performance characteristics needed for the intended applications. The DQ documentation also serves as a reference point for verification tests carried out during the Operational Qualification (OQ) stage.  

Installation Qualification (IQ) confirms that the instrument or equipment has been delivered as per the approved design and specifications. It also ensures that the system is correctly installed in the intended location and that the installation environment is appropriate for its intended operation and use.  

Operational Qualification (OQ) is the step where the instrument is tested to confirm that it operates as intended under the chosen conditions. It ensures that the HPLC system meets the essential functional and operational criteria defined during the Design Qualification (DQ).  

Performance Qualification (PQ) involves confirming that the instrument delivers results reliably and in line with the specifications required for its routine applications. The emphasis is on verifying that the instrument performs consistently over time. 

Documentation 

After completing equipment qualification, the following records should be maintained: 

• Design Qualification (DQ) report 
• Installation Qualification (IQ) report, including details of hardware and software 
• Standard procedures for Operational Qualification (OQ) testing 
• Test reports generated during OQ 
• Performance Qualification (PQ) protocols along with representative test results. 

High Performance Liquid Chromatography (HPLC) 

 High Performance Liquid Chromatography (HPLC) is a highly sophisticated and flexible analytical technique utilized for the separation, identification, and quantitative determination of components in complex mixtures. The process involves the passage of a liquid sample through a column packed with a stationary phase under elevated pressure. Variations in the interactions of analytes with the stationary phase enable their separation according to distinct physicochemical characteristics. Due to its superior sensitivity, resolution, and reliability, HPLC has become an indispensable tool in diverse fields such as pharmaceutical quality control, environmental monitoring, food analysis, and clinical research. 

Principle  

The fundamental principle of HPLC lies in the differential distribution of analytes between a stationary phase and a mobile phase. The stationary phase generally comprises finely divided, porous particles contained within a column, while the mobile phase consists of one or more solvents propelled at high pressure. The sample is introduced into the mobile phase stream through an injection valve and sample loop, allowing it to enter the column. Based on their affinity toward the stationary phase, individual components migrate at different rates, resulting in effective separation. Upon elution, the analytes are detected by a suitable detector, and the signals are processed through system software to generate a chromatogram. This chromatogram provides both qualitative and quantitative information regarding the separated constituents. 

Types of HPLC  

HPLC techniques can be broadly categorized in two ways

According to the scale of operation:  

Preparative HPLC – primarily used for the isolation and purification of compounds in larger quantities. 

Analytical HPLC – employed for qualitative and quantitative analysis of samples 

According to the separation principle:  

• Affinity chromatography Adsorption chromatography, 
• Size-exclusion chromatography 
• Ion-exchange chromatography 
• Chiral phase chromatography   

Based on the elution technique:  

Isocratic separation – employs a mobile phase of constant composition throughout the run. Gradient separation – involves a gradual change in the composition of the mobile phase to improve separation efficiency. 

Based on the mode of operation:  

 Normal phase chromatography – utilizes a polar stationary phase and a non-polar mobile phase. 

 Reverse-phase chromatography – employs a non-polar stationary phase with a relatively polar mobile phase. 

Normal Phase Chromatography: 

Normal Phase HPLC (NP-HPLC) separates compounds on the basis of polarity. It uses a polar stationary phase combined with a non-polar mobile phase. Polar analytes interact more strongly with the stationary phase, resulting in greater retention. As the polarity of an analyte increases, adsorption forces strengthen, causing a longer elution time. 

Reversed Phase Chromatography:  

Reversed Phase HPLC (RP-HPLC or RPC) employs a non-polar stationary phase and a moderately polar or aqueous mobile phase. The separation mechanism relies on hydrophobic interactions, where non-polar analytes are retained due to their affinity toward the hydrophobic stationary phase. The extent of retention is proportional to the size and surface area of the analyte’s non-polar groups in contact with the stationary phase. 

• Instrumentation of HPLC:  

Figure 1. Instrumentation of HPLC

The development and validation of analytical methods are crucial in the research, formulation, and large-scale production of pharmaceutical dosage forms. These processes ensure the identity, purity, potency, safety, effectiveness, and overall performance of medicines. In cases where no official methods are available, new analytical procedures are established to meet the requirements of novel drug products. For non-pharmacopoeial substances, alternative methods are often created to reduce cost, save time, and enhance accuracy and robustness. Whenever an alternative technique is proposed to replace an existing one, comparative data must be presented to demonstrate its benefits and potential limitations. The primary aim of HPLC method development is to obtain efficient separation and precise quantification of the active pharmaceutical ingredient (API), impurities, intermediates, and degradation products. 

Essential Steps in HPLC Method Development  

Assessment of Physicochemical Properties 

Evaluate solubility, stability, molecular weight, pKa, and other parameters that influence chromatographic performance. 

Selection of Chromatographic Conditions 

Choose the suitable stationary phase, mobile phase, column type, and detection technique based on the drug’s properties.

Defining the Analytical Strategy 

Establish clear objectives such as resolution, sensitivity, reproducibility, and acceptable run time. 

Sample Preparation 

Develop optimized procedures for sample preparation to ensure reproducibility, minimize interferences, and achieve reliable results. 

Optimization of Method Parameters 

Adjust critical factors including flow rate, column temperature, gradient conditions, and injection volume to improve separation efficiency and overall method performance. 

Method Validation: 

Establish the reliability of the developed method by systematically evaluating its accuracy, precision, specificity, and robustness through a structured validation process. 

? Applications of HPLC  

Purification: 

Purification through HPLC involves isolating a specific compound from a mixture containing structurally similar substances or impurities. Under optimized chromatographic conditions, each compound produces a unique peak. To achieve successful purification, the analyst must carefully choose suitable mobile and stationary phases according to the physicochemical properties of the compounds, ensuring efficient separation and recovery of the desired analyte during elution. 

Chemical Separation: 

HPLC is widely applied for chemical separation by utilizing differences in compound interactions with the stationary phase and their mobility in the mobile phase. This approach enables the resolution of structurally related molecules, including enantiomers. The effectiveness of separation depends largely on the appropriate choice of chromatographic phases, making HPLC a powerful tool for differentiating compounds within complex mixtures. Identification: 

One of the major uses of HPLC is compound identification and assay. Analytical parameters are adjusted to ensure that the chromatographic peak of the test sample corresponds to that of the reference standard. At the detection limits employed, the peak must be well-defined, properly labelled, and distinctly resolved from other signals, allowing reliable identification. 

Other Applications: 

Beyond pharmaceutical analysis, HPLC has extensive applications across multiple fields. It is employed in clinical diagnostics, forensic investigations, food and environmental monitoring, and various research domains where accurate separation, quantification, and characterization of compounds are essential. 

Introduction Of Spectroscopy  

Spectroscopy refers to the study of how matter interacts with electromagnetic radiation. During these interactions, matter can either absorb or emit energy in the form of radiation. Broadly, spectroscopy is categorized into two types: absorption and emission spectroscopy. Absorption spectroscopy—such as UV-Visible, Infrared (IR), Nuclear Magnetic Resonance (NMR), microwave, and radiofrequency spectroscopy—focuses on analysing the specific wavelengths of electromagnetic radiation absorbed by a sample.

Ultraviolet (UV) Spectroscopy  

Ultraviolet (UV) spectroscopy, commonly referred to as UV-Visible (UV-Vis) spectrophotometry, is based on the measurement of light absorption within the ultraviolet and visible regions of the electromagnetic spectrum. This method is widely adopted across scientific disciplines owing to its simplicity, cost-effectiveness, and versatility. For a compound to be analysed, it must contain a chromophore— a functional group capable of absorbing radiation in the UV-Vis range. 

UV-Visible spectrophotometry is extensively applied for both qualitative and quantitative analysis of compounds in diverse samples. The principle involves passing a beam of light through the sample and detecting absorbance at selected wavelengths. The extent of absorption is proportional to the concentration of the absorbing species, forming the basis for quantitative determinations. 

In addition, this technique serves as a complementary tool to fluorescence spectroscopy. Key analytical parameters evaluated include absorbance (A), transmittance (%T), and reflectance (%R), along with monitoring their variations over time. 

Terms used in spectroscopy   

A. Chromophore 

• In Greek word: chromo means colour and phores means bearer 
• A Chromophore is the part of molecules responsible for its colour. The colour that is seen by our eyes. 
• Define as any isolated covalently bonded group that shows a characteristic absorption of electromagnetic radiation in the UV or visible region. 
• A group that gives rise to absorption in visible and near ultra violet is called "chromophore" 
• There is a faint absorption band between 200 and 300 nm for compounds with nonconjugated carbonyl groups.

B. Auxochrome: 

• In Greek: Auxo means increase and chromo means colour 
• Auxochrome is defined as the any group of which does not itself act as a chromophore but whose presence brings about the shift of the absorption bands towards the red end of the spectrum, (longer wavelength). 
• The effect is due to its ability to extent the conjugation of a chromophore by sharing the non- bonding electrons. 
• Take benzene, phenol, and aniline as examples; their Amax values are (255 nm), (270 mm), and (280 nm). 

Fig 2. The electromagnetic spectrum with the visible light section expanded

? Principle of UV-Visible Spectroscopy:  

Beer’s Law forms the theoretical basis of UV–Visible spectroscopy. It states that when a beam of electromagnetic radiation passes through an absorbing medium, the transmitted intensity (I) is lower than the incident intensity (I?). The absorbance (A) is quantitatively related to the concentration (c) of the absorbing species and the path length (b) of the sample cell as: A = εLc 

where ε represents the molar absorptivity (L·mol?¹·cm?¹). This direct proportionality enables UV–Visible spectroscopy to be a powerful tool for quantitative analysis. 

When molecules absorb UV or visible light, electrons are promoted from lower-energy orbitals to higher-energy orbitals. This process involves transitions between bonding, non-bonding, and antibonding orbitals. 

Ground state orbitals: 

1. σ (bonding orbital) 
2. π (bonding orbital) 
3. n (non-bonding orbital) 

Antibonding orbitals:  σ* (sigma antibonding orbital )

1. π* (pi antibonding orbital)  

It is noteworthy that non-bonding (n) electrons do not participate in bond formation; hence, no corresponding n antibonding orbital exists. As a result, only specific electronic transitions are possible upon absorption of ultraviolet or visible radiation. These include: 

1. σ → σ* (sigma to sigma star) 
2. n → σ* (non-bonding to sigma star) 
3. n → π* (non-bonding to pi star) 
4. π → π* (pi to pi star) 

Fig 3. Electronic Transition graphically represented

Fig 4. Instrumentation Of UV-Visible Spectrophotometer

1. Filters and Monochromators 
2. Sample cell 
3. Detectors 
4. Recording Devices 

1.Sources of Radiation  

The ideal light source for spectroscopic applications should provide high stability, strong intensity, and a broad emission range, typically from 180–700 nm. 

Hydrogen Discharge Lamp 

This lamp contains hydrogen gas maintained under high pressure. When subjected to an electric discharge, the excited hydrogen molecules emit radiation in the ultraviolet region.

Deuterium Lamp 

Structurally similar to the hydrogen discharge lamp, this source uses deuterium gas instead of hydrogen. It provides higher intensity, approximately three to five times greater than hydrogen lamps, making it more efficient for UV applications.

 Xenon Discharge Lamp 

In this type, xenon gas is pressurized between 10–30 atm and placed between two tungsten electrodes. It offers significantly greater intensity compared to hydrogen lamps. 

Mercury Arc Lamp 

This lamp operates using mercury vapours. However, due to its discontinuous spectrum, it is less favoured and rarely used as a reliable source of radiation. 

2. Filters and Monochromators  

The main function of these devices is to separate a broad band of polychromatic radiation into a narrower band of nearly monochromatic radiation. 

? Filters 

Filters are employed to isolate a specific, narrow range of radiant energy from the overall spectrum. They selectively transmit radiation within the desired wavelength region while absorbing most of the unwanted wavelengths. 

Types of Filters: 

Glass filters 

Gelatine filters 

Interference filters 

? Prisms 

Prisms, generally fabricated from materials such as glass, quartz, or fused silica, serve as dispersing components in spectrophotometers. Among these, glass exhibits approximately three times greater dispersing power than quartz, making it particularly suitable for use in the visible region of the spectrum. The ability of a prism to separate polychromatic radiation into narrow wavelength bands arises from the variation in refractive index with wavelength. 

Fig 5. Prism

Cornu Type 

Littrow Type 

? Diffraction Grating 

A higher degree of spectral dispersion can be achieved using diffraction gratings. These consist of a large number of finely ruled, parallel grooves—typically ranging from 15,000 to 30,000 lines per inch—on a highly polished aluminium surface. The grooves act as scattering centers for the incident light beam, producing diffraction. The resolving power of a grating is directly related to the number of lines per unit length, with greater line density resulting in enhanced resolution. 

3. Sample Cells  

Sample containers, commonly referred to as cells or cuvettes, are designed with optically transparent windows suitable for the spectral range under investigation. Quartz or fused silica cuvettes are essential for ultraviolet measurements (below 350 nm) but can also be used in the visible region. For wavelengths between 375–2000 nm, silicate glass is preferred due to its lower cost compared to quartz. Plastic cuvettes are also employed for measurements in the visible region. 

For optimal performance, the cell windows should be oriented perpendicular to the incident light beam to minimize reflection losses. The standard path length for UV-visible spectroscopy is 1 cm, with matched and calibrated cuvettes of this dimension being readily available from commercial suppliers.

Fig 6. Sample Cells

4. Detectors  

Detectors used in UV–visible spectrophotometers are generally referred to as photometric detectors. The most widely employed types include: 

1. Barrier layer cell (photovoltaic cell) 
2. Phototube (photo-emissive tube) 
3. Photomultiplier tube 

Barrier Layer Cell (Photovoltaic Cell): 

Also called a photonic cell, this detector operates without an external power source. It is composed of a metallic base plate, usually made of iron or aluminium, which serves as one electrode. A thin layer of a semiconductor material such as selenium is coated on this base. The selenium surface is further covered with a very thin film of silver or gold, functioning as the second collector electrode. 

Fig 7. Barrier Layer Cell

A phototube consists of an evacuated glass tube that houses a photocathode and a collector anode. The photocathode is coated with materials of high atomic weight, such as caesium, potassium, or silver oxide, which emit electrons upon exposure to light. These emitted electrons are attracted to the anode, generating a current directly proportional to the incident light intensity. To enhance performance, composite coatings like caesium/caesium oxide/silver oxide are often employed, extending sensitivity and operational range, particularly in the UV region. Additionally, the output signal can be amplified using external circuits. Compared to photovoltaic cells, phototubes offer greater sensitivity, making them more commonly utilized in spectrophotometric applications. 

Fig 8. Phototube (Photo-Emissive Tube)

Photomultiplier tubes are highly sensitive detectors, commonly integrated into advanced instruments such as spectrofluorometers, due to their ability to measure extremely low light intensities. Their working principle is based on the multiplication of photoelectrons through secondary electron emission. This is achieved using a photocathode in combination with a cascade of anodes, known as dynodes, typically arranged in a series of up to ten. Each dynode is maintained at a potential difference of about 75–100 V higher than the preceding one, resulting in progressive amplification of the electron signal. 

Owing to this mechanism, PMTs are capable of detecting signals up to 200 times weaker than those measurable with photovoltaic cells, making them invaluable in fluorescence-based measurements. However, to ensure accuracy, they must be adequately shielded from stray light interference.

Fig 9. Photo-multiplier tube