Calibration of Analytical Instruments

Set to operate within the necessary range, the primary purpose of calibration is quality assurance, and each pharmaceutical instrument needs to have its accuracy tested regularly. ICH states that the act of comparing an instrument or device's output with that of the reference or traceable standard throughout a suitable range of measurement to demonstrate that it generates findings within predetermined bounds is known as calibration. In the pharmaceutical business, fast and accurate instrument calibration is crucial to guaranteeing the quality of a pharmaceutical product's manufacturing process. Before generating products for the market, calibration documentation is required. For optimal use, an instrument must be calibrated correctly, aiding in the accuracy of the instrument and adjusting it to the desired and appropriate precision for the given use and necessity. Calibration involves calibrating the equipment to measure the required range of values if it is outside of that range. This is the primary justification for why calibration, especially calibration of instruments, is crucial. Pharmaceutical companies also value calibration. Current calibration or regression software, whether instrument-based or posterior analysis, uses ordinary least squares as an estimate of the calibration parameters. In this paper, we compare the default or normal (ordinal least squares) regression approach applied to trace metal data. Lack of proper curve-fitting methodology. Ultracentrifugation (AUC) is a traditional technique used to measure the macroscopic redistribution of particles in a solution, allowing for the assessment of hydrodynamic and thermodynamic properties across a wide range of sizes (Svedberg and Pedersen 1940; et al. 2015). Being label-free and requiring only the initial sample without any additional matrix, AUC can be applied to a variety of substances ranging from small molecules to proteins, nucleic acids, carbohydrates, as well as synthetic polymers, nanoparticles, supramolecular complexes, and even whole organisms. Its core principle involves the application of a precisely defined centrifugal force on particles in a gravitational field, with sedimentation velocity (SV) being the most utilized AUC mode in recent years, primarily due to its ability to provide dynamic measurements of particle movement. While linearity is a fundamental feature of valid analytic measurement systems, the term itself is not specifically mentioned in CLIA. Instead, CLIA refers to three closely associated concepts: calibration, calibration verification, and reportable range. [1,3]

1. Types of calibration: -

The nature, type and purpose of the measurement to be made will determine the type of calibration required.

• 1. Direct Calibration
  2. Indirect Calibration
• Direct calibration:
  • The response of the measuring device and the value of the standard are both expressed in the same quantity.
  • Units of measurement are used to express the standard measurement indicator and the analytical balance indicator.[2]

• Indirect Calibration: -
  • Another name for it is analytical Calibration.
  • The standard value and the equipment answer have different magnitudes. The values that the measuring apparatus produces, known as signals or instrumental responses, differ from the properties of standards in terms of amount and are expressed in disparate units. A The result of the known measurement for each standard is used to determine the instrumental response, which is obtained with an indirect calibration technique
  • Two examples of indirect calibration are calibrations with spectrophotometers and chromatographs. The height of the peak or the area of the peak and the concentration of the standard solution are related to the calibration of the chromatograph. The calibration spectrophotometer determines the correlation between the optical type (absorption, emission, wavelength, etc.) and the concentration of the standard solution. [2]

1. 3. Need for calibration:

• • A novel tool.
  • Following maintenance or modification, an instrument.
  • Once a predetermined amount of time has passed.
  • When a given period of time (the operation hours) has passed.
  • Prior to, during, or following a crucial measurement.
  • Following an incident, for instance.
  • When a shock, vibration, or unfavorable situation has been experienced by an equipment that may have caused it to become misaligned, or hurt it.
  • Unexpected variation in the climate.
  • If there are any doubtful observations or mismatched instrument indications, output from substitute instruments.
  • As required, e.g., by a customer specification or the recommendation of the instrument maker.
  • When used generally, calibration is commonly understood to include the process of modifying a measurement instrument's output or indication to agree with the applied standard’s value, inside a given level of precision. An example of this would be calibrating a thermometer to find the indication error or correction and then adjusting it (for example, using calibration constant) so that it displays that accurate temperature in Celsius at given the scale points. This is how the final users of the instrument see it. But few instruments can be precisely tuned to match the criteria by which they are judged. Regarding the enormous, in most cases, the calibration procedure is truly a comparison between an unknown and an acknowledged standard, documenting the outcomes. [3]

1. 4. Calibration Standard:

Accredited companies use ISO 9001 as a quality benchmark for calibrating measurements. This process involves documenting each stage of the intricate operation. ISO 9001 is a quality management standard applicable to businesses in various industries.

For calibration, laboratories use the ISO/IEC 17025 quality standard, where IEC refers to the International Electrotechnical Commission. This standard ensures that the results are valid and align with the established values. [1]

1. 5. Importance of Instrument Calibration:

For effective use, instruments must be calibrated correctly. This aids in analyzing the accuracy of the instrument and adjusting it to the desired and appropriate precision for the given use and necessity. Calibration involves calibrating the equipment to measure the required range of values if it is outside of that range. This is the primary justification for why calibration—especially calibration of instruments—is crucial. When the precision of the instrument has an impact on the end product, it is crucial. Pharmaceutical companies also recognize the value of calibration.

When measuring the thickness of a wire, for instance, a screw gauge that has not been calibrated correctly would display zero error, which needs to be adjusted or corrected for in order to obtain an exact measurement. In the pharmaceutical sector, calibration is the same. The industry produces drugs that actively affect our immune systems, so the calibration procedure needs to be done with the utmost caution. When compared to the expense of calibration, the costs associated with using an uncelebrated instrument are exponential.

To examine the effects and requirements for concentration of the various pharmaceutical products, biological, chemical, or biochemical analysis can be performed. It is crucial to make sure that the instruments are calibrated appropriately for the purpose when utilizing other devices to analyses these items. [1]

1. 6. Application of Calibration:

Calibration is used in a wide range of applications in electronics, including

• 6. 1. Calibration is used in a wide range of applications in electronics, including:1. Calibration is used in manufacturing processes to maintain quality control throughout the production process and make sure that electronic devices and systems are generating accurate and consistent outputs. Scientific and medical research
     2. To guarantee that electronic instruments and systems used in research produce correct and trustworthy data, calibration is necessary.
     3. Environmental Monitoring: Used to confirm that electrical devices and sensors that detect temperature, pressure, humidity, and other variables produce accurate readings.
     4. English: Aerospace and Defense: In order to guarantee that electronic systems and equipment satisfy industry standards and regulations and generate accurate and trustworthy information, calibration is essential in the aerospace and defense sectors.
     5. Electricity generation and distribution: In the energy sector, calibration verifies that the electronic devices and systems used in electricity generating, transmission, and distribution give accurate and trustworthy measurements while adhering to rules and industry standards.
     6. Consumer electronics: Additionally, calibration is carried out [4]
  7. Advantages of Calibration:
• Save money: Profits will rise as costs are reduced, and waste is eliminated through standardization.
• Ensure Safety: Products and parts that have been calibrated operate as intended and are safe to use.
• Ensure Quality: Reliable production of standardized goods is possible with calibrated machinery and components.
• Shorten Production Time: Quality components save time when they are available to replace tolerance components.

Assure Compliance to Certification: It is guaranteed that all industry rules and business certifications will be followed.

• Determine Accuracy: Calibration will enable accurate product reporting.
• Prevent and predict process failure: Steady progress towards process conditions beyond tolerance can be detected using sensors that are accurate and dependable.[1]
  8. Disadvantages of Calibration:
• The primary drawback of calibration could be a loss of environmental control. Sensitive electronics may be affected if, for example, the room where the equipment is located cannot be properly controlled for temperature and humidity. [1]
  9. Calibration Of Pharmaceutical Instrument:

• • UV Visible Spectrophotometer
  • IR – Spectrophotometer
  • Fluorimeter
  • Flame photometry
  • High performance liquid chromatography (HPLC)
  • Gas Chromatography

• UV Visible Spectrophotometer:

UV visible spectroscopy, often known as UV spectroscopy, is the study of absorption or reflectance in portions of the electromagnetic spectrum that fall between the complete, nearby visible region and the ultraviolet. This analytical method calculates how much of each discrete UV or visible wavelength is absorbed or transmitted through the sample relative to the reference or blank sample. The sample mixture has an impact on the properties, providing information about the composition and concentration of the sample, perhaps. The UV spectrum is approximately between 100 to 450 nm, and the visible spectrum is approximately between 450 to 700 nm.[5]

• • Calibration process of UV-visible spectroscopy:
  • Ensure the power cable of the instrument is properly plugged into the socket and the cuvettes are clear.
  • Turn on the device.
  • Allow 15 minutes for it to warm up.

Keep the dummy cuvette in the sample holder's position.[1].

•     Control of absorbance:

Testing the absorbance at the UV wavelengths with a potassium dichromate solution yields the precise value of X (1%, 1cm) for each wavelength, as well as the allowable limits of a given absorbance. +0.01 is the absorption tolerance. Using potassium dichromate solution that has been UV dried to a constant weight at 130 degrees Celsius, 57.0 to 63.0 mg of potassium dichromate UV should be dissolved in 0.005 M sulfuric acid and then diluted to 1000 ml using the same acid in order to control the absorbance in between approximately 235 nm to 313 nm.[1]

•     Limit of stray light:

•     When comparing the absorbance of a 1.2% w/v potassium chloride solution in a 1 cm cell to water as a reference liquid, for instance, the absorbance of the solution should be more than 2.0 at approximately 200mm in order to detect stray light at the specified wavelength.[1]

•     Resolution Power:

When we specified in a monograph, note the UV spectrum of a 0.02% v/v toluene in hexane solution. Unless the monograph specifies otherwise, the ratio of the absorbance at the maximum is approximately 269 nm, to that at the minimum approximately 256 nm, is not less than 1.5. [1]

•     Holmium Perchlorate Solution:

•     Melt 0.5 g of holmium oxide in 2.4 ml of perchloric acid, then dilute to 10 ml of H2O to obtain a solution of holmium per chlorate.

•     Take note of the absorption spectra is approximately between 230 to500 nm.

• • Take note of the Holmium perchlorate solution's spectra is approximately between 200 to 600 nm utilizing 1.4 M of perchloric acid as the benchmark.
  • Record the wavelength maxima in relation to the approval criteria. [1]
  • Holmium Filter:
  • The Holmium filter works well for routine calibrations. The narrowest slit setting and slowest scan speed should be used to capture the absorption spectra is approximately between 490 to 250 nm. Recognizing one band at around 360.5 millimeters and three fused absorption bands centered at 452.2 nm.[1]

     
           
       
 Fig 2: UV Visible Spectrophotometer [7]

• • Low-pressure discharge lights are suitable.
  • Pay attention to the mercury lamp's transmission spectrum, ranging from approximately 590 to 230 nm, beside the monochromator's entrance. Utilize the narrowest slit setting and the slowest scan speed achievable.

Mercury's primary emission lines are positioned at mm 579.0, 576.5, 435.8, 364.9, and 253.7.[1]

• IR Spectrophotometer:
  • When infrared radiation passes through a sample, an infrared spectrophotometer measures the relative energy as a function of the infrared radiation’s wavelength or frequency. An analytical tool for material identification, particularly organic polymer identification, is the IR spectrophotometer. When infrared radiation passes through a sample, an infrared spectrophotometer measures the relative energy as a function
  • Of the infrared radiation’s wavelength or frequency. IR spectrophotometry studies. chemical compounds or functional groups by measuring the interaction of infrared radiation with matter. Two types having an IR range of 22800–10 cm is dispersive infrared (DIR) and Fourier transform infrared (FTIR).
  • The range of IR spectrophotometer is approximately between 2500 to 25000 nm.

•     Make sure the platform where the FT-IR equipment is mounted is clear of dust and clean.

•     Verify that the power supply is connected to the instrument.

•     Make that the units’ communication cords are connected correctly.

•     Checks for the dry indicator should be made before the instrument is turned on. On the front, there are two indicators: a green lamp for electricity and an orange lamp for dryness. Even when it is turned off, the FT-IR interferometer’s dry indicator light indicates the humidity within

•     Make sure the dry indication is on at all times.

•     Prior to activating the device, the silica gel must also be checked.

•     Checking the power spectrum. To do background scan measurement, click the (BKG Scan) icon. A power spectrum that was seen following scarving.

•     After selecting the data tab, adjust the scan parameters, such as the number of scans and resolution.

•     Resolution Tests for IR Spectrophotometer:

•     To make it easier to compare with published reference spectra, certain pharmacopoeias prescribe Resolution Tests.

•     These tests calculate the trough depth between two partially resolved peaks.

•     This means that, in addition to the width at half height of isolated bands, which serves as the foundation for published instrument specifications, the results also depend on the geometry of the lines.

•     Due to variations in default apodization function selections, spectrometers from different manufacturers may have varied line forms for the same nominal resolution.

•     Despite the fact that variations don’t really matter for material identification, they have the power to determine whether a resolution test is passed or not.

•     For instance, the band measures 1601 cm in width and half height, with a resolution of 4 cm.

•     Although the depth of the trough at 1583 cm is reduced by 12%, this is increased by less than 0.3 cm when the apodization is changed from mild Norton-Beer to strong Norton- Beer.[1]

The wavelength range of a fluorimeter is approximately between 210 to 890nm.

• • • As stated in the MEASURE SETUP menu's CALIBRATION SETUP, make sure the proper cuvette(s), blank solution, and standard solutions are readily available before starting the calibration. Choose the necessary technique by following the instructions in the "Using a stored method" section.
    • To start the calibration procedure, press the CAL key once when in measure mode. Place the cuvette with the blank solution, being careful not to mark the optical surfaces in the roomy chamber's cuvette holder and shut the lid.
    • Press the CAL key or use the CAL option to measure the fluorescence of the blank attempt, displayed as highlighted on the instrument display. To verify, use the enter key.
    • Throughout the measurement, the status message "Reading" will appear on the instrument screen.
    • After finishing, a big number will appear on the screen representing the concentration zero, since this is the blank. The percentages of full-scale emission and the raw fluorescence for the signal and reference are shown below this.

There will also be changes to the options at the bottom of the screen. The calibration phase that was just finished is related to the ACCEPT and REPEAT options. Choose ACCEPT to proceed through the calibration sequence to the first standard if the concentration and other calibration data are accurate and satisfactory. Choose REPEAT and repeat the previous step if the data from the previous stage were.

unacceptable. By using ABORT, the instrument can be brought back to the measure screen and the entire calibration sequence can be cancelled.

• • • When the blank calibration is accepted, the instrument will display the message "Please insert standard xxx" along with the first standard concentration specified in the Measure/Calibration Setup's Standards Table.
    • Close the sample chamber lid after removing the Blank solution cuvette and replacing it with the required standard cuvette. Use appropriate cleaning techniques if you are using reusable cuvettes to ensure no solutions are carried over. Removal of impurities
    • Use the CAL key or the on-screen CAL option to calibrate the instrument with this initial standard, just as you did with the blank.
    • Like the blank, the standard concentration (as specified in the STANDARD TABLE) will be shown on the screen along with the raw fluorescence and the full-scale emission percentages for the reference and signal.
    • Review this data again, and if it's acceptable, select the ACCEPT option to proceed to any more calibration standards in the same way.
    • This method of calibrating with each standard will be followed (points 10-15) until all established standards have been met. At this point, the device will display the raw fluorescence for each standard in a table to provide an overview of the calibration. A plot of the standards' concentration against their raw fluorescence can be seen at the bottom of the screen to observe the calibration curve.
    • The instrument reverts to the measurement screen and status after the calibration summary message "Cal needed" has been eliminated.

Now, samples with unknown concentrations can be measured.[1]

• The flame photometer’s principle relies on measuring the intensity of light released upon the addition of a metal to the flame. Information about the element is provided by the colour’s wavelength, and information about the quantity of the element in the sample is provided by the flame’s colour. An analytical tool for measuring calcium, lithium, sodium, and potassium ions in bodily fluids is a flame photometer, which is used in clinical laboratories.[1]

The wavelength range of flame photometer is approximately between 210 to 790nm.

When anything such as sodium or potassium is aspirated into a flame, an optical device known as a flame photometer is utilized to gauge the intensity of the color. The aim of the flame photometer measuring process is to measure the emission light of the sample.

1)   Compressor Readings: When the instrument's compressors were read to determine whether the involved pressure was within the typical range (14–30 psi) as stated by the manufacturer, the investigation discovered that 80% of the compressor was operating within the limit and 20% was operating above it. The pressure gauge regulates 20% of the compressor's pressure.

2)   The Pressure Gauge Accuracy: In order to determine if the pressure gauges are accurate and if the regulated pressure is within the normal range as defined by the manufacturer, the pressure gauge control is monitored both before and after calibration using OPI in psi units. According to the examination, 90% of the pressure gauges were in good condition; the remaining 10% need replacement.

3)   The sucking rate of the nebulizer: According to the manufacturer's instructions, the nebulizer's sucking rate should be between two and six milliliters per minute. High values of the sucking rate before calibration cause significant differences between pre and post calibration rates, as shown by paired samples T-test. Flame photometry's concept is that, for various concentrations, light released from the flame is directly linked to the inhaled species' concentration.

• To ensure standard and sample backgrounds match, a calibration curve is created using a standard solution with known component concentrations. Additional material may be added as necessary. The expected sample concentration determines the calibration curve's coverage, allowing readings to fall mid-curve.
• After plotting the calibration curve, sample concentration is determined by comparing values with the curve. Each element has a unique calibration curve, so separate curves must be created. Calibrating once and regular checks suffice for consistent estimations. Recalibrating an instrument is easy; set the top standard to match the initial calibration curve value and the blank solution to read zero.[1]

• HPLC (High Performance Liquid Chromatography)

• High Performance Liquid Chromatography, or HPLC for short, is the method used to separate the constituents of a liquid medium. Solvent (mobile phase) passes through a separation medium-packed column (stationary phase).
• High-performance liquid chromatography analysis of pharmaceuticals is used to track the effectiveness of a disease’s treatment, verify a drug’s identity, and offer quantitative data. [1]

The wavelength range for HPLC is approximately between 180 to 900nm.