Viva Preparation for Quality Control Department
Table of Content:
* Abbreviations
* Instruments
* CRS Management
* WS Management
* Reagent Management
* Water Analysis
* HPLC/UPLC/GC Column Management
* Instrument Calibration
* Glassware Calibration
* Titration
* Stability
* Raw Material Sampling
* Packaging Material
* Differences
* Acceptance Criteria for retest
* TS
* VS
* Calculation
Abbreviation
* ATR = Attenuated Total Reflectance
*EDQM = European Directorate for the Quality of Medicines
* MSDS = Material Safety Data Sheet
Instruments
* pH meter
* Conductivity Meter
* HPLC
* UV Spectophotometer
* IR
* FTIR
* AAS
* Particle size Analyzer
* Karl Fisher Titrator
* Potentiometer
* Osmometer
* Viscometer
* TOC Machine
* Dissolution Tester
pH meter
Why 3M Potassium Chloride is used in pH meter?
Answer: 3M Potassium chloride solution is used in pH meters primarity to maintain the hydration of the glass membrane of the pH electrode and to prevent unwanted ion exchange. This helps ensure stable and accurate pH measurements.
Conductivity Meter
HPLC System
Differences between Gradient and Isocratic Elution
Detectors used in HPLC
☞ UV visible Detectors
☞ Fluorescence Detector
☞ Refractive Index Detector
☞ Mass Spectrometry
Fluoresvence Detector: A fluorescence detector can measure fluorescence in the wavelength range of 220-90 nm. Since the fluorescence wavelength depends on the excitation wavelengrh, the results are more selective than can be obtained using a UV/vis detector.
Procedure to switch over HPLC instrument from Reverse phase to.Normal Phase:
☞ Wash the HPLC system with water to remove residues of buffers from mobile phase lines, pump, auro sampler, detector and needly wash.
☞ Wash the HPLC system including lines, pump, auto sampler, detector and needle wash with Isopropyl Alcohol.
☞ Run the mobile phase of normal phase in the HPLC system.
Basic Principle of HPLC
The fundamental principle of High-Performance Liquid Chromatography (HPLC) is separation based on differential interactions between sample components and a stationary phase within a column, while using a mobile phase (liquid solvent) to carry the sample through. This process relies on the concept of partitioning, where compounds in the sample distribute themselves between the stationary and mobile phases.
Types of HPLC
There areabout four types of HPLC . These are:
1) Normal Phase HPLC
2) Reverse Phase HPLC
3) Size Excusion Chromatography
4) Ion Exchange Chromatography
Parts of HPLC
1) Solvent/Mobile Phase Reservoir
2) Pump
3) Injector
4) Column
5) Detector
Normal Phase HPLC
Normal-phase HPLC (NP-HPLC) is a chromatographic technique that uses a polar stationary phase and a less polar mobile phase to separate compounds based on their polarity. In contrast to reverse-phase HPLC, where the stationary phase is nonpolar and the mobile phase is polar, NP-HPLC separates compounds where more polar compounds interact more strongly with the polar stationary phase and elute later.
Key aspects of Normal-Phase HPLC:
Stationary Phase: Typically polar, like silica gel or alumina.
Mobile Phase: Less polar, using solvents like hexane, heptane, dichloromethane, chloroform, or ethyl acetate.
Separation Principle: Compounds are separated based on their relative affinity for the polar stationary phase and the nonpolar mobile phase. More polar compounds will be retained longer by the stationary phase, while less polar compounds will elute more quickly.
Applications: Suitable for separating lipophilic compounds, long-chain alkanes, compounds that are sparingly soluble in water, and phospholipids.
Advantages: Can be particularly useful for separating isomers, which can be difficult to separate with other HPLC techniques.
Considerations: Reproducibility can be affected by water adsorption on the silica surface, and solvent demixing can occur with gradient elution.
Reverse Phase HPLC
Reverse phase high-performance liquid chromatography (RP-HPLC) is a common analytical technique that separates compounds based on their hydrophobicity (or polarity). It uses a non-polar stationary phase and a polar mobile phase, which is usually a mixture of water and an organic solvent. This technique is widely used for analyzing and purifying small molecules, especially in biomedical and pharmaceutical applications.
Separation Principle:
In RP-HPLC, the stationary phase is typically a non-polar material, like silica beads coated with long alkyl chains (e.g., C18). The mobile phase is a mixture of water and a polar organic solvent (like acetonitrile or methanol). Hydrophobic molecules in the sample tend to interact more strongly with the non-polar stationary phase and are retained longer. More polar molecules will be eluted more quickly by the mobile phase.
Total Line of HPLC-7 (For Shimadzu)
1. Line-A: Purified Water
2. Line-B: Solvent (Methanol)
3. Line-C: Acetonitrile
4. Line-D: Mobile Phase (+Buffer)
5. Rinse Line (50% Acetonitrile + 10% IPA)
6. Waste Lines (2 lines)
Pre-wash Procedure of HPLC Column (Prior to Analysis/Mobile Phase Run)
1) Set the desired coloumn on column compartment of HPLC.
2) Purge every line with purified water for at least 2 minutes.
3) Depending on the organic phase composition in mobile phase, gradually reach the same. For example, if 30% organic phase (Acetonitrile/methanol/Others) used in mobile phase and storing solvent for column is 97% organic phase (Acetonitrile/methanol/Others), then starting with 97% organic phase (Acetonitrile/methanol/Others), gradually decrease the organic phase and finally reach 30%.
4) For gradient flow, thus can be conducted through time program and for Isocratic flow, this caan be done using intermediate solvent, as appropriate. Accomplish this run within 15-20 minutes.
Post-wash Procedure of HPLC Column (After Accomplishing Analysis/Mobile Phase Run) for the mobile phase containing Buffer and Counter ion:
1) Replace the mobile phase with purified water. Run the same for 15-20 minutes.
2) Depending on the storing solvent, gradually reach the same. For example, if 97% Organic phase
(Acetonitrile/methanol/Others) is storing solvent, then starting with 95% purified water, gradually increase the organic phase and finally reach at 97%.
3) For the gradient flow this can be conducted through time program and for isocratic flow, this can be done using Intermediate solvents like 15%, 30%, 455 Organic phase as appropriate.
4) Accomplish this run within 55-60 minutes conducting each solvent composition period 15-20 minutes.
Post-wash Procedure of HPLC Column (After Accomplishing Analysis/Mobile Phase Run) for the mobile phase without Buffer and Counter ion:
2) Depending on the storing solvent, gradually reach the same from the organic phase composition of mobile phase. For example, if 97% Organic phase (Acetonitrile/methanol/Others) is storing solvent and organic phase composition in mobile phase is 30%, then starting with the initial organic phase (Acetonitrile/methanol/Others) of 30% (70% Purified water), gradually increase the organic phase and finally reach at 97%.
3) For the gradient flow this can be conducted through time program and for isocratic flow, this can be done using Intermediate solvents like 15%, 30%, 455 organic phase as appropriate.
4) Accomplish this run within 55-60 minutes conducting each solvent composition period 15-20 minutes.
Post-wash Procedure of HPLC Column (After Accomplishing Analysis/Mobile Phase Run) for the mobile phase containing Tri-ethyl Amine:
1) Perform the initial wash with 90% Washing Solvent (a mixture of Methanol: 0.05% Phosphoric act at 50:50 ratio) and 10% organic solvent.
2) Run the mixture for 30 minutes.
3) Depending on the storing solvent, gradually reach the same. For example, if 97% Organic phase
(Acetonitrile/methanol/Others) is storing solvent, then starting with 20-30% organic phase and gradually increase the organic phase and finally reach at 97%.
4) For the gradient flow this can be conducted through time program and for isocratic flow, this can be done using Intermediate solvents like 15%, 30%, 455 organic phase as appropriate.
5) Accomplish this run within 55-60 minutes conducting each solvent composition period 15-20 minutes.
Buffer for HPLC
In HPLC, a buffer solution is a component of the mobile phase used to maintain a stable pH. The mobile phase itself is the liquid that carries the sample through the HPLC column. While the mobile phase can be composed of various solvents, including water and organic solvents, a buffer solution is often added to the mobile phase to control and stabilize the pH.
Mobile Phase:
The mobile phase in HPLC is the liquid used to carry the sample through the chromatographic column. It's typically a mixture of water and an organic solvent, such as acetonitrile or methanol.
Buffer Solution:
A buffer solution is a mixture that resists changes in pH when small amounts of acid or base are added. In HPLC, buffers are commonly used to stabilize the pH of the mobile phase, ensuring consistent separation of analytes.
Relationship:
Buffers are often used in the mobile phase to help maintain the desired pH. For example, if adding a sample to the mobile phase might shift the pH, a buffer can be added to resist those changes.
Importance of pH Control:
The pH of the mobile phase can significantly affect the retention and separation of different analytes, especially when dealing with ionizable compounds. By controlling the pH with a buffer, researchers can optimize the separation process and achieve reliable results.
Examples:
Common buffers used in HPLC include phosphate buffers, acetate buffers, and ammonium buffers.
In summary, a buffer solution is a component of the mobile phase designed to control and stabilize the pH, while the mobile phase itself is the liquid that carries the sample through the HPLC column.
What will be the problem if pH of Mobile Phase will higher than acceptable limit?
A higher pH in the mobile phase, particularly beyond certain limits, can lead to several problems in chromatography, primarily affecting the retention and stability of the column. Specifically, increasing the pH can cause acidic compounds to become more ionized and less retained, while basic compounds become more retained as they deprotonate. Furthermore, high pH can lead to premature column failure due to silica dissolution in silica-based columns, especially above pH 8.
Here's a more detailed breakdown:
1. Retention Changes:
Acidic Compounds:
At higher pH, acidic compounds are more likely to lose a proton (H+), becoming ionized (negatively charged). This makes them more polar and less likely to interact with the hydrophobic stationary phase, leading to reduced retention and shorter retention times.
Basic Compounds:
Conversely, at higher pH, basic compounds are more likely to gain a proton, becoming neutral or positively charged. This makes them less polar and more likely to interact with the stationary phase, leading to increased retention and longer retention times.
2. Column Degradation:
Silica Dissolution:
High pH can dissolve the silica support used in many chromatographic columns, especially in reversed-phase chromatography. This can lead to column damage and a reduced lifetime.
Bonded Phase Degradation:
The bonded phase (the stationary phase) can also degrade at high pH, further impacting column performance and lifespan.
3. Method Robustness:
Peak Broadening:
Changes in pH can affect the symmetry and width of chromatographic peaks, leading to peak broadening or tailing.
Selectivity Issues:
If the mobile phase pH is too close to the pKa of the analytes, small pH changes can have a significant impact on separation selectivity, making it difficult to separate closely eluting peaks.
4. Buffer Issues:
Buffer Stability:
Each buffer has a specific pH range where it provides optimal stability. Using a buffer outside its optimal range can lead to pH drift and inaccurate results.
Precipitation:
In some cases, using high pH buffers or solutions can lead to buffer precipitation in the HPLC system, causing blockages and damaging the system.
What will be the problem if pH of Mobile Phase will lower than acceptable limit?
If the pH of the mobile phase in liquid chromatography falls below the acceptable limit, it can lead to several issues, including column damage, altered retention times, and poor peak shapes. Specifically, acidic conditions can damage the stationary phase (silica-based columns) if the pH is too low, potentially dissolving the silica or causing bonded phase hydrolysis. Furthermore, changes in pH can significantly affect the ionization and retention of ionizable analytes, leading to shifts in retention times and potentially impacting peak tailing or broadening.
Here's a more detailed breakdown:
1. Column Damage:
Silica-based columns:
Silica, the material commonly used in reversed-phase columns, is sensitive to acidic conditions. If the pH of the mobile phase is too low (below 2), it can lead to the silica dissolving or the bonded phase being cleaved from the silica, effectively damaging the column and shortening its lifespan.
Bonded phase hydrolysis:
Acidic conditions can also cause the bonded phase (the layer attached to the silica) to hydrolyze, which can further damage the column and alter its performance.
2. Altered Retention Times:
Ionizable analytes:
The pH of the mobile phase significantly influences the ionization state of ionizable analytes (acids and bases). Changes in pH can shift the equilibrium of these compounds, affecting their retention in the column.
Retention time shifts:
A small change in pH (as little as 0.1 units) can lead to noticeable shifts in retention times, which can be problematic for quantitative analysis and method development.
3. Impact on Peak Shapes:
Peak tailing:
Acidic conditions can interact with residual silanol groups on the silica surface, leading to unwanted interactions with basic analytes, which can result in peak tailing.
Peak broadening:
In reversed-phase chromatography, if the pH is too low, the analyte's interactions with the stationary phase may not be as strong as desired, potentially leading to peak broadening.
4. Other Considerations:
Buffer selection:
Using a buffer in the mobile phase helps to maintain a stable pH and minimize fluctuations, which can further reduce the risk of column damage and ensure consistent retention times, says hplc.eu.
Selectivity:
Mobile phase pH can also be a tool for manipulating the selectivity of the separation, allowing analysts to optimize the separation of closely related compounds by adjusting the pH to influence the interactions between analytes and the stationary phase.
In summary, maintaining the mobile phase pH within an acceptable range is crucial for ensuring column stability, accurate retention times, and optimal peak shapes in liquid chromatography. Careful consideration of the analyte's ionization behavior, the column's pH limits, and the use of buffers are essential for successful chromatographic separations.
Procedure to switch over HPLC instrument from Normal Phase to Reverse Phase:
☞ Wash the HPLC system with Isopropyl Alcohol to remove residues of buffers from mobile phase lines, pump, auro sampler, detector and needly wash.
☞ Wash the HPLC system including lines, pump, auto sampler, detector and needle wash with Water.
☞ Run the mobile phase of Reverse phase in the HPLC system.
Standard Reproducibility
The reproducibility of Standard solution-A and Standard solution-B must be between 98.0% and 102.0% (in case of Assay,Dissolution and Conten Uniformity)
Standard Reproducibility = (Average area of Std-B/Average area of Std-A) × (Weight of Std-A/Weight of Std-B) × 100
Handling of System Suitability Failures:
☞ If system suitability parameters are not meeting the acceptance criteria then aise LIR also investigate.
Examples of Failures:
☞ High Pressure
☞ Column Leakage
☞ Communication failure between system & CDS.
☞ System is stopped qhile gradient is running
☞ %RSD for duplicate sample injections is more than %RSD mentionex for system suitability standards.
☞ Retention Time variation/Shift
☞ Unstable base line/humps or noise/Electronic spikes
☞ Parially acquired Chromatograms.
Processing and reporting of the chromatograms shall not exceed more than 3 working days from the date of acquisition of injection in the sample set.
System Suitability
System suitability refers to a set of criteria used to assess the performance of an analytical system, such as a chromatographic system, before it's used to analyze samples. It ensures the system is operating correctly and can provide reliable results.
System suitability Parameters of HPLC
| Parameter | Acceptance Criteria | Remarks |
|---|---|---|
| Resolution | >2 (For Separation of two peaks) | Ensures adequate separation between peaks for accurate quantification of individual analytes. A higher resolution indicates better separation. |
| Repeatability | less than 1% for peak area and retention time, for 5 replicates | Measures the precision of the method. A low RSD indicates good reproducibility and accuracy. RSD is often calculated as the standard deviation of the peak response divided by the mean response, multiplied by 100%. |
| Column Efficiency (Theoretical Plates, N) | > 2000 (minimum, depends on column length and dimensions) | Reflects the efficiency of the column in separating the analytes. A higher number of theoretical plates generally indicates better separation. |
| Tailing Factor (T) | less than 2 (ideally less than 1.5) | Measures the peak shape. A tailing factor close to 1 indicates a symmetrical peak, while higher values indicate peak tailing, which can affect accurate quantification. |
| Retention Time | Within specified limits (established during method development) | Ensures that the analytes elute within the expected time frame. Retention time is affected by factors like mobile phase composition, flow rate, and column type. |
| Capacity Factor (K) | > 2 (minimum, depends on the analyte and method) | Relates the time a compound spends in the stationary phase (column) to the time it spends in the mobile phase. A higher capacity factor generally indicates a longer retention time. |
| Signal-to-Noise Ratio (S/N) | > 10 (minimum, depends on the analyte and method) | Indicates the strength of the signal relative to the background noise. A higher S/N ratio ensures reliable detection of the analytes. |
| Specificity | Peaks of diluent, placebo, and impurities do not interfere with the peaks of the analyte. | Ensures that the method can specifically detect the analyte of interest without interference from other compounds in the sample. |
| Purity | Peaks of the analyte are pure. | Indicates that the analyte is not co-eluting with any other components. |
| Peak Purity factor | ≥ 0.9995 (minimum). | Ensures that the analyte peak is pure and free from co-eluting impurities. |
| Relative Standard Deviation (RSD) | RSD ≤ 2% for peak area and retention time | Measures precision (repeatability). |
What is Baseline Noise?
Baseline noise in HPLC refers to the random fluctuations or wiggles observed in the baseline of a chromatogram. These fluctuations can be caused by various factors, including instrumental issues, mobile phase impurities, column problems, and environmental factors. A noisy baseline can make it difficult to accurately identify and quantify analytes, especially those present at low concentrations.
Signal-to-Noise Ratio
In High-Performance Liquid Chromatography (HPLC), the signal-to-noise ratio (S/N) is a crucial parameter that indicates the sensitivity of the method and is used to determine the limit of detection (LOD) and limit of quantification (LOQ). A higher S/N indicates a stronger signal relative to the background noise, resulting in better sensitivity and more accurate results.
Here's a more detailed explanation:
What is S/N?
S/N is the ratio of the peak signal (the peak height or area) to the baseline noise (the fluctuation of the baseline).
It's a measure of how well the signal of the analyte of interest stands out against the background noise.
A high S/N indicates a strong signal, while a low S/N indicates a weak signal that may be obscured by noise.
How S/N is Used in HPLC:
Determining LOD and LOQ:
S/N is a key factor in determining the minimum concentration of an analyte that can be detected (LOD) and quantified (LOQ) in a sample.
Generally, an S/N of 3:1 is considered the LOD, while an S/N of 10:1 is considered the LOQ.
SNR between 3:1 and 10:1 for LOD
SNR from 10:1 to 20:1 for LOQ
Method Validation:
S/N is used in method validation to assess the sensitivity and precision of the HPLC method.
Troubleshooting:
A low S/N can indicate problems with the instrument, sample preparation, or chromatographic conditions, according to an article on Chromatography Online.
How to Calculate S/N:
S/N is calculated by dividing the peak signal by the noise.
The peak signal is typically measured as the peak height from the baseline to the peak's maximum.
The baseline noise is the fluctuation of the baseline in a blank run (a run with no analyte).
Some instruments and software automatically calculate S/N, according to a Chromatography Forum thread.
Improving S/N:
Optimize Chromatographic Conditions:
Adjusting mobile phase composition, flow rate, and column temperature can improve peak shape and reduce baseline noise.
Improve Sample Preparation:
Ensuring proper sample extraction and cleanup can reduce the amount of interfering compounds and improve S/N.
Use More Sensitive Detectors:
Some detectors, such as mass spectrometers (MS) and diode array detectors (DAD), can provide higher sensitivity and better S/N compared to traditional UV detectors.
Adjust Instrument Settings:
Optimizing detector gain, signal amplification, and time constants can also improve S/N.
What do you mean RSD?
Relative standard deviation (RSD), also known as the coefficient of variation (CV), is a statistical measure that expresses the standard deviation of a dataset as a percentage of its mean. It's essentially a way to understand how much data points vary around the average, relative to that average. A higher RSD indicates greater variability, while a lower RSD suggests more consistency or precision.
Purpose: RSD helps quantify the spread or dispersion of data points within a dataset, especially when comparing datasets with different means or units.
Calculation: The formula for RSD is (standard deviation / mean) * 100.
Units: RSD is expressed as a percentage.
Interpretation: A lower RSD means the data points are more tightly clustered around the mean, indicating higher precision or consistency. Conversely, a higher RSD means the data points are more dispersed, suggesting less precision or consistency.
Which types of Buffer are more commonly used in HPLC?
In HPLC, phosphate buffers and acetate buffers are frequently used. Phosphate buffers are particularly common for separating acidic compounds, while acetate buffers are often preferred for separating basic compounds. Other common buffers include citrate and ammonium acetate, often used in specific applications like LC-MS.
Phosphate Buffers:
These are popular because they are easy to prepare, offer a wide pH range, and have low UV absorption, which is important for UV detection. They are also stable when mixed with organic solvents and don't react with sample compounds or degrade the column.
Acetate Buffers:
These are also widely used, especially in applications where a slightly lower pH range is needed compared to phosphate buffers. They are often preferred for separating basic compounds.
Other Buffers:
Citrate Buffers: These can be used for separating metal ions.
Ammonium Acetate: This is a volatile buffer, making it compatible with LC-MS, where the mass spectrometer requires volatile compounds to be analyzed.
Trifluoroacetic acid (TFA): A common volatile buffer, also used in LC-MS.
Formic Acid: Another volatile buffer commonly used in LC-MS applications.
Buffer Selection Factors:
The choice of buffer depends on several factors, including:
pH range: The pH range of the buffer should match the pH required for separation of the desired compounds.
UV absorption: The buffer should have low UV absorbance at the detection wavelength.
Compatibility with LC-MS: If LC-MS is being used, volatile buffers are preferred.
Sample properties: The properties of the samples being analyzed will influence the choice of buffer.
How to Prepare Phosphate Buffer SOlution for HPLC?
To prepare a phosphate buffer for HPLC, you'll typically mix a solution of sodium dihydrogen phosphate (NaH₂PO₄) and a solution of disodium hydrogen phosphate (Na₂HPO₄) to achieve the desired pH and concentration. For example, to make a 0.2 M buffer at pH 7.4, you would combine 81 mL of a 0.2 M Na₂HPO₄ solution with 19 mL of a 0.2 M NaH₂PO₄ solution. You'll also need to filter the final solution to remove any insoluble particles.
Here's a more detailed breakdown:
1. Prepare Stock Solutions:
Na₂HPO₄ (Disodium Hydrogen Phosphate):
Dissolve 2.84 g of Na₂HPO₄ (anhydrous) in distilled water to make 1 L of a 20 mmol/L solution.
NaH₂PO₄ (Sodium Dihydrogen Phosphate):
Dissolve 2.40 g of NaH₂PO₄ (anhydrous) in distilled water to make 1 L of a 20 mmol/L solution.
2. Mix to Achieve Desired pH:
Combine the stock solutions to reach the target pH. For a pH of 7, you would mix equal volumes of the 20 mmol/L solutions of both Na₂HPO₄ and NaH₂PO₄.
Note: The exact ratio of Na₂HPO₄ and NaH₂PO₄ will depend on the desired pH. You can adjust the ratio based on the buffer's pKa value and the desired pH.
3. Adjust to Final Volume and Concentration:
After mixing, dilute the solution with distilled water to the desired final volume and concentration.
For example, if you want a 0.2 M buffer, you can dilute the combined solution to a total volume of 100 mL.
4. Filtration:
Filter the solution through a 0.45 μm or smaller pore size filter to remove any insoluble particles. This prevents clogging of the HPLC pump and column.
Important Considerations for HPLC:
Solubility:
Ensure that the buffer is completely soluble in the chosen organic solvent (e.g., methanol, acetonitrile) used in your mobile phase. If the buffer precipitates when mixed with the organic solvent, it can cause problems with the HPLC system.
Concentration:
Choose a buffer concentration that is compatible with your HPLC method and the solubility of the buffer in the mobile phase.
pH:
Select a pH that is appropriate for your analysis and the stability of the analytes being separated.
Mobile Phase Compatibility:
Ensure that the buffer is compatible with the chosen organic solvent and detector used in your HPLC system.
By following these steps, you can prepare a phosphate buffer suitable for use in HPLC
UV Spectrophotometer
Basic Principle of UV Spectroscopy:
UV spectroscopy measures the absorbanve of ultraviolet and visible light by a substance, providing information about its molecular structure and concentration. The principle involves exciting electrons in molecules to higher energy levels when they abdorb specific wavelengths of UV or Visible light.
Spectrophotometry
Spectrophotometry is a technique that uses light absorption to measure the concentration of an analyte solution.
Beer Law:
A beam of visible light passing through the solution is proportional to the solution concentration.
Lambert Law:
Lambert law states that absorbance and path length is directly proportional.
When a beam of monochromatic light is passex through a pure homogenous absorbing medium, the rate of decrease in intensity of a radiation with the thickness of absorbing medium is proportional to the intensity of incident light.
Beer-Lambert Law:
The Beer-Lambert law is a linear relationship between the absorbance and the concentration, molar absorpyion coefficient and optical path length of a solution.
The amount of light absorbed by a solution is related to the analyte concentration which is expressed as follows: A =£bc
Where, £ is tge molar absorptivity of the analyte, b is the path lengtg (the distance the light travels through the solution) and c is the concentration of the analyte.
Wavelength range of UV spectroscopy:
☞ UV spectroscopy typically covers a wavelength range from 190 to 400 nm., with visible range fron approximately 400 to 800 nm.
Visible wavelength range:
Violet: 400-420 nm
Indigo: 420-440 nm
Blue: 440-490 nm
Green: 490-570 nm
Yellow: 570-585 nm
Orange: 585-620 nm
Red: 620-780 nm.
Which compound absorb UV wavelength?
Compound with conjugated pi syatems, particularly those with multiple double bonds, are likely to absorb UV light in UV-visible spectroscopy. These compounds, also known as chromophore, exhibit a shift in absorption to longer wavelength as the conjugation increases.
N.B: The more extended the conjugation, the longer the wavelength of light absorbed.
IR & FTIR
Principle of IR Spectroscopy
Infrared (IR) spectroscopy is based on the principle that molecules absorb specific frequencies of infrared light, which are characteristic of their vibrational and rotational motions. This absorption leads to changes in the energy levels of these motions, revealing information about the molecule's structure and bonding.
Molecular Vibrations:
Molecules are not static entities; their atoms are constantly vibrating around their equilibrium positions. These vibrations are governed by the strength and geometry of the chemical bonds.
Infrared Absorption:
When molecules are exposed to infrared radiation, certain frequencies of light are absorbed if they match the energy required to excite specific vibrational modes.
Spectral Analysis:
The absorbed infrared radiation results in a spectrum, a plot of intensity versus wavelength (or frequency).
Structural Information:
The positions and intensities of the absorption bands in the spectrum provide information about the types of bonds present in the molecule and its overall structure.
Functional Group Identification:
The presence of certain functional groups, such as C=O, C-H, and N-H, will be indicated by specific absorption bands in the infrared spectrum.
Purity and Identity:
Infrared spectroscopy can also be used to assess the purity of a compound and to identify unknown substances.
Types of Vibrations in IR Spectroscopy
In IR spectroscopy, molecules vibrate in two primary ways: stretching and bending. Stretching involves changes in bond length (either symmetric or asymmetric), while bending involves changes in bond angles. Further, bending vibrations can be categorized into scissoring, rocking, wagging, and twisting.
Elaboration:
Stretching Vibrations:
These vibrations involve changes in the distance between two atoms within a molecule, essentially "stretching" and "compressing" the bond. Stretching vibrations are mainly two types: Symmetric vibration and Asymmetric Vibration.
Symmetric Stretching: Both atoms involved in the bond move in the same direction, either towards each other or away from each other.
Asymmetric Stretching: One atom moves towards the other, while the other moves in the opposite direction.
Bending Vibrations:
These vibrations involve changes in the angle between two bonds.
Scissoring: Atoms on either side of a bond move in a scissor-like motion, changing the angle.
Rocking: Atoms on either side of a bond move in a rocking motion, back and forth.
Wagging: A group of atoms attached to a central carbon moves up and down, perpendicular to the C-H bond.
Twisting: A group of atoms attached to a central carbon moves in a twisting motion around the C-H bond.
In essence: IR spectroscopy analyzes the absorption of infrared light by molecules, revealing information about these vibrational modes, which can be used to identify functional groups and understand molecular structure.
IR Wavelength Range
Infrared (IR) radiation spans wavelengths roughly from 750 nanometers (nm) to 1 millimeter (mm). This range can be further divided into sub-regions: near-infrared (NIR, 780 nm - 2.5 µm), mid-infrared (2.5 - 50 µm), and far-infrared (50 - 1000 µm). IR waves have longer wavelengths than visible light but shorter than radio waves.
Elaboration:
Wavelength Range:
The infrared region of the electromagnetic spectrum is defined as encompassing wavelengths between approximately 750 nm and 1 mm.
Sub-regions:
The IR range is often divided into three sub-regions:
Near-infrared (NIR): Typically ranges from 780 nm to about 2.5 micrometers (2.5 µm).
Mid-infrared (MIR): Generally spans from 2.5 µm to about 50 µm.
Far-infrared (FIR): Covers wavelengths from 50 µm to 1,000 µm (1 mm).
Frequency:
Infrared radiation has frequencies higher than microwaves but lower than visible light frequencies, ranging from approximately 300 GHz to 400 THz.
Comparison to Visible Light:
Infrared waves are longer than visible light waves, meaning they have lower frequencies.
Wavenumer Range of FTIR
FTIR spectra typically cover the mid-infrared region, which corresponds to a wavenumber range of approximately 4000 to 400 cm⁻¹ (or wavelengths from 2.5 to 25 micrometers). This range allows for the identification and quantification of various materials by measuring their absorption of mid-infrared light.
Here's a more detailed breakdown:
Mid-infrared region: This is the primary area of interest for FTIR spectroscopy, offering a rich spectral fingerprint for various molecules.
Wavenumber range: 4000 cm⁻¹ to 400 cm⁻¹.
Wavelength range: 2.5 to 25 micrometers.
Purpose: FTIR analysis in this range allows scientists to study the vibrational modes of molecules, providing information about the chemical structure and functional groups present in a sample.
Key features: The position and intensity of absorption peaks in the spectrum can be used to identify specific bonds and functional groups within a molecule.
How FTIR measure functional group of a compound?
FTIR spectroscopy identifies functional groups by measuring a compound's absorption of infrared light at specific frequencies, which corresponds to the vibrations of the functional groups. These vibrations are characteristic of particular bonds within the molecule, allowing scientists to identify and even quantify the presence of certain functional groups.
Here's a more detailed explanation:
1. Infrared Light and Molecular Vibrations:
Infrared (IR) radiation has energy that can excite molecules to vibrate, and the frequencies at which these vibrations occur are related to the types of bonds within the molecule.
2. Absorption and the FTIR Spectrum:
When IR light passes through a sample, specific wavelengths are absorbed by the functional groups, resulting in a spectrum with peaks at those wavelengths.
3. Functional Group Identification:
The peaks in the spectrum correspond to characteristic frequencies of different functional groups. For example, a peak at 1730 cm⁻¹ might indicate a carbonyl (C=O) group, while a peak at 3300 cm⁻¹ could suggest an O-H or N-H bond.
4. Quantitative Analysis:
The intensity of the peaks can be used to estimate the concentration of a functional group in the sample.
5. Distinguishing Between Similar Groups:
While FTIR can identify the presence of specific functional groups, it may be challenging to differentiate between closely related groups, like ketones and carboxylic acids, according to the UC Davis Air Quality Research Center.
6. Fingerprint Region:
The fingerprint region of the spectrum, typically between 1500 and 400 cm⁻¹, contains many peaks that are unique to the specific molecule and can be used for further identification.
In essence, FTIR spectroscopy leverages the unique vibrational characteristics of functional groups to provide insights into the molecular structure and composition of a compound.
Various Wavenumbers used in FTIR for Compound identification
FTIR spectroscopy uses specific wavenumbers to identify functional groups and, ultimately, compounds. Wavenumbers, expressed in cm-1, are the units most commonly used to identify bands in FTIR spectroscopy. By analyzing the spectrum, it's possible to determine which functional groups are present, which can help identify the compound.
Key Wavenumber Ranges and Associated Functional Groups:
* 3000-3300 cm-1: Typically associated with O-H and N-H stretching vibrations.
* 2900-3000 cm-1: Often indicates C-H stretching in alkanes, methyl, methylene, and methyne groups.
* 1700-1750 cm-1: Suggests the presence of C=O (carbonyl) groups, such as in aldehydes, ketones, and esters.
* 1600-1650 cm-1: May indicate C=C stretching in alkenes, or aromatic compounds.
* 1100-1400 cm-1: Typically associated with various C-O and C-N stretching vibrations, as well as fingerprint region skeletal vibrations.
* Fingerprint Region (below 1500 cm-1): This region is highly specific to a particular compound and can be used to identify it.
900-920 cm-1: Can indicate Ge-O-Alδ vibrations.
790-820 cm-1: Can also indicate Ge-O-Alδ vibrations.
683-695 cm-1: Shows various vibrations, particularly Al-O.
550-590 cm-1: Shows various vibrations, particularly Al-O.
How FTIR is Used for Compound Identification:
1. Sample Preparation:
The compound is prepared in a suitable form for analysis, such as a solution or a solid pellet.
2. Spectra Acquisition:
The FTIR spectrometer scans the sample and generates an infrared spectrum, which is a plot of the absorption of infrared light as a function of wavenumber.
3. Spectral Analysis:
The spectrum is analyzed to identify the wavenumbers of the absorption bands.
4. Functional Group Identification:
The presence of characteristic bands at specific wavenumbers allows the identification of functional groups.
5. Compound Identification:
By comparing the spectrum to known spectral databases or literature data, the compound can be identified.
What is finger Print Region?
The fingerprint region in infrared (IR) spectroscopy is the wavenumber range from about 1500 to 500 cm⁻¹. It is called the "fingerprint" region because the absorption pattern in this area is highly unique for each molecule, much like a human fingerprint.
Differences between IR and FTIR
Infrared (IR) and Fourier Transform Infrared (FTIR) spectroscopy are both methods for studying the vibrational frequencies of molecules, but FTIR is a more advanced technique that offers several advantages over traditional IR spectroscopy. FTIR is faster, more sensitive, and provides higher resolution data due to its ability to collect all wavelengths of IR light simultaneously using interferometry.
Key Differences:
Speed:
FTIR is significantly faster as it collects data across the entire spectrum simultaneously, while traditional IR spectroscopy scans one wavelength at a time.
Sensitivity:
FTIR is more sensitive due to its ability to acquire more data in less time, making it suitable for analyzing low concentration samples.
Resolution:
FTIR generally provides higher resolution spectra, allowing for more detailed analysis of molecular structures.
Data Acquisition:
FTIR uses interferometry to obtain an interferogram, which is then converted to a spectrum using a Fourier transform. Traditional IR spectroscopy directly measures the intensity of transmitted or absorbed IR light.
Instrument Complexity:
FTIR instruments are more complex than traditional IR spectrometers, involving interferometers and advanced optical systems.
In essence, FTIR is a more advanced and versatile technique that offers advantages in speed, sensitivity, and resolution compared to traditional IR spectroscopy, making it the preferred choice for many applications.
Interferometer
In Fourier Transform Infrared (FTIR) spectroscopy, an interferometer, specifically a Michelson interferometer, is the core component that manipulates the infrared light to generate a spectrum. It splits the IR beam into two paths, reflects them off mirrors, and then recombines them. The interference pattern created by the recombined beams contains information about the different wavelengths present in the original IR radiation.
Fourier Transform
In FTIR spectroscopy, the Fourier transform is a mathematical process that converts a complex signal, collected in the time domain, into a spectrum that reveals the frequencies of infrared radiation absorbed or emitted by a sample. This allows for the identification of functional groups and compounds within the sample.
Atomic Absorption Spectroscopy (AAS)
Basic Principle of AAS
The fundamental principle of Atomic Absorption Spectrometry (AAS) is that atoms in a sample absorb light at specific wavelength and the amount of light absorbed is directly proportional to the concentration of the element being measured. This absorption occurs when electrons in the ground state of an atoms are exited to higher energy levels by absorbing photons of light at characteristic wavelengths.
Gas Chromatography
Chromatography is a technique that separates components in a mixture by the difference in partioning behavior between mobile and stationary phase. Gas Chromatography is one of the popular chromatography techniques to separate volatile compounds or substances. The mobile phase is a gas such as helium and the stationary phase is highgboiling liquid that is absorbed on a solid.
Principle of Gas Chromatography
The principle of gas chromatography is partition. The mixture of component to be seoarated is converted to vapour and mixed wit gssrous mobile phase. The component which is more soluble in stationary phase travel slower and elutes later. The component which is less soluble in stationary phase travels faster and eluter out first. No two components has same partition coefficient conditions. So the components are separated according to thrir partition coefficient.
Partition coefficient is the ratio of solubility of a substances distributed between two immiscible liquids at a constant temperature.
How does gas chromatography work?
First, the sampl is introduced into a stream of inert gas or a carrier, which is usually helium or argon. For a liquid sample, it needs to be eveporatex before being injected into the carrier. Sample components move through the packed column at,a rate affected by the degree of interaction of each component with stationary non-volstile phase. Substances that interact more with the stationary phase are delayed and thus separated from substances and interact less. When the components are eluted from the coumn, they can be quantified andmor collected through the detector for further analysis.
Detectors used in Gas Chromatography:
☞ Flame Ionization Detector (FID)
☞ Thermal Conductivity Detector (TCD)
☞Electron Capture Detector (ECD)
☞ Mass Spevtrometer (0MS)
TOC Machine
How to TOC Machine measure the amount of TOC in water?
TOC machine measures the amount of organic carbon in a water sample by oxidizing the organic matter to Carbon dioxide (CO2) and then quantifying the resulting CO2. This process typically involes three main steps: Oxidation, detection and calculation.
Dissolution Tester
Dissolution Apparatus-1 (Basket Apparatus):
Temperature: 37 ± 0.5°C
Acceptance criteria for Immediate Release Dosage forms:
| Stage | Number Tested | Acceptance Criteria |
|---|---|---|
| S1 | 6 | Each unit is not less than Q + 5% |
| S2 | 12 | Average of 12 unite is equal to or greater than Q and no unit is less than Q -15% |
| S3 | 24 | Average of 24 units is equal to or greater than Q, no more than 2 units are less than Q-15% and no unit is less than Q-25% |
Dissolution Medium
☞ If the dissolution medium is buffered solution, adjust the solution so that the pH is within 0.05 unit of specified pH.
Apparatus Setting
The distance between the inside bottom of the vessel and the basket/blsde to be maintained at 25±2 mm during the test.
Time:
Time tolerane = ±2%
Chemical Reference Substance (CRS)
What is INN CRS?
Chemical Reference Substance (CRS): Chemical Reference Substances are authentic substances that have been approved by Reference Standard approving authority as suitable for use as comparision in various tests described in individual monograph and method analysis.
☞ For USP RS, Consider potency as 100% unless potency is specified in the respective CRS container label and for BP/EP CRS follow potency specified in the respective leaflet or on-line catalogue.
☞ For BP/EP CRS, if the potency is not mentioned for the active substanves, then these CRS can only be used for qualitative analysis.
☞ When use an impurity reference standard in a related substances test for the control of an impurity and if no assigned content is indicated, the purity of the reference standard for the purpose of estination is considered to be 100%.
☞ For non-pharmacopeial CRS, use potency referring to the respective certificate.
☞ For USP RS, in some cases Previous lot might be mentioned with a validity date in the list, in this case, same can be used.
☞ For EP CRS current lot is valid for all sub lot/batches.
☞ If any CRS is excluded from "Current lot" list, then place a fresh requisition for the same. Until receiving of the same, use the existing CRS. Upon receiving of the current lot of CRS, discard the old batch of CRS using internal requisition and issue in IFS software.
☞ Check the current lot status monthly (Preferably 1st week of the every month) and update the CRS list.
☞ Purchase requisition should be raised for INN CRS before about six months of their expiry date and for USP, EP and BP CRS before about four months of their expiry date using UFS software.
Expiry dating
If the batch/Lot number on the BP CRS vial is different from the current batch/Lot on the website, then apply an expiry date of 12 months from the date of dispatch provided if it is un-opened and stored according to the instruction given.
Use & Discard of CRS
☞ Store and use the prepared CRS solution used for qualitative test at 2°C to 8°C for three months, unless otherwise specified in the individual monography/method.
☞ Discard INN CRS immediately after their expiry date and use the Stock USP, EP and BP CRS until receiving of fresh Lot/Batch of RS and CRS. The old batch of RS and CRS will be discarded after receiving of the current lot of RS and CRS.
Working Standards (WS)
Reagent Management
☞ In case of expiry date, if expiry date related information is not provided in manufacturer COA, give 5 years expiry of the reagents from the date of receiving.
☞ After opening of the reagent re-assign the expiry date as foolws:
⇨ For solid Reagent (Non-hygroscopic, Non-degradable, Non-sensitive): Expiry date is 3 years after opening.
⇨ For solid Reagent (hygroscopic, degradable, sensitive): Expiry date is 2 years after opening.
⇨ For Solvent/Liquid (Non-hygroscopic, Non-degradable, Non-sensitive): Expiry date is 3 years after opening.
Note: After opening revised expiry date should not exceed Manufacturer recommended expiry date or initial expiry date after receiving.
Raising purchase requisition
☞ Raise the requisitionconsidering three months stock in hand for local purchase and six months stocks in hand for LC purchase.
☞ For local purchase, safety stock shall be three months stock and LC purchase it is six months.
Water Analysis
Types of Water
☞ Potable Water
☞ Purified Water
☞ Water for Injection
☞ Fresh water
☞ Salt water
☞, Brackish water
☞ Hard Water
☞ Soft Water
☞ Distilled water
☞ Waste water
☞ Black water
☞ Grey Water
☞ Raw water
Acceptance Criteria of Various types of Water:
Purified Water:
| Test Parameters | Accepatance Criteria | Method Reference |
|---|---|---|
| Description | Clear, colorless, Odorless liquid | USP 43 |
| pH | 5.00–7.00 | USP 43 |
| Conductivity | NMT 1.30 at 25°C and 1.10 at 20°C | USP 43 |
| TOC | NMT 500 ppb | USP 43 |
| Nitrates | NMT 0.2 ppm | BP 2023/Ph.Eur 11.0 |
| Total Viable Count (TVC) | NMT 100 CFU/mL | USP-43 |
Water for Injection (WFI)
| Test Parameters | Accepatance Criteria | Method Reference |
|---|---|---|
| Description | Clear, colorless, Odorless liquid | USP 43 |
| pH | 5.00–7.00 | USP 43 |
| Conductivity | NMT 1.30 at 25°C and 1.10 at 20°C | USP 43 |
| TOC | NMT 500 ppb | USP 43 |
| Nitrates | NMT 0.2 ppm | BP 2022/Ph.Eur 10.0 |
| Total Viable Count (TVC) | NMT 10 CFU/100 mL | USP-43 |
| Bacterial Endotoxin Test | NMT 0.25 EU/mL | USP-43 |
Potable Water:
| Test Parameters | Accepatance Criteria | Method Reference |
|---|---|---|
| Description | Clear, colorless, Odorless liquid | In house |
| pH | 6.50–8.50 | In house |
| Conductivity | NMT 250 uS/cm | In-house |
| Total Hardness | NMT 500 mg/L | In-house |
| Heavy metal | NMT 0.1 mg/L | In-house |
| Iron | NMT 0.3 mg/L | In-house |
| Arsenic | NMT 10 ppb | In-house |
| Free Chlorine | NMT 0.4 mg/L | In-house |
| Total Viable Count (TVC) | NMT 500 CFU/mL | USP-43 |
Effluent Treatment Plant Water (ETP Water)
| Test Parameters | Acceptance Criteria | Reference Method |
|---|---|---|
| Description | Visual Inspection | In-house |
| pH | 6.00 to 9.00 | ECR-2023 |
| Total Dissolved Solid (TDS) | NMT 2100.0 mg/L | ECR-2023 |
| Dissolve Oxygen (DO) | 4.5 to 8.0 mg/L | In-house |
| Chemical.Oxygen Demand (COD) | NMT 200 mg/L | ECR-2023 |
| Biological Oxygen Demand (BOD) | NMT 30 mg/L | ECR-2023 |
| Total Suspended Solid | NMT 100 mg/L | ECR-2023 |
What is DO in water?
Dissolved oxygen (DO) in water refers to the amount of oxygen gas (O2) that is dissolved in water and available for aquatic life. It's a crucial factor for the health of aquatic ecosystems and is a key indicator of water quality.
What will be happen if DO levels become higher than Acceptable Limit?
If water's dissolved oxygen (DO) levels become excessively high, it can be detrimental to aquatic life and potentially lead to other water quality issues. High DO levels can cause gas bubble disease in fish, and it can also contribute to stress or death in other organisms.
Here's a more detailed explanation:
Gas Bubble Disease:
When DO levels are too high, it can force oxygen into the bloodstream of fish, forming bubbles that can obstruct blood flow, leading to gas bubble disease and potentially death.
Stress and Impaired Function:
While not as severe as gas bubble disease, excessively high DO can also stress aquatic organisms and potentially impair their normal biological functions.
Impact on Other Organisms:
High DO can affect other aquatic organisms, including invertebrates and microorganisms, potentially disrupting the delicate balance of the ecosystem.
Other Water Quality Issues:
High DO can sometimes be a symptom of other underlying water quality problems, such as excessive algal blooms or changes in temperature, which can further impact aquatic life.
In summary, while high DO levels are generally not as harmful as low DO levels, they can still pose risks to aquatic ecosystems and potentially cause stress or death in some organisms.
What will be happen if DO levels become higher than Acceptable Limit?
If Dissolved Oxygen (DO) levels fall below acceptable limits, it can lead to severe consequences for aquatic life, including fish and other aquatic organisms. Low DO levels can cause stress, reduce growth, impair reproduction, and even lead to widespread fish kills.
Here's a more detailed look at the effects:
Stress and Reduced Growth:
When DO levels are too low, fish experience stress and may stop eating, making them more vulnerable to diseases.
Reproductive Issues:
Sensitive species, like salmon, may struggle to reproduce at low DO levels.
Fish Kills:
If DO levels drop significantly, especially below 3 mg/L, fish can suffocate and die.
Changes in Aquatic Communities:
Lower DO levels can favor certain species that are more tolerant of low oxygen conditions, leading to shifts in the overall structure of the aquatic community.
Increased Activity of Anaerobic Bacteria:
Low oxygen environments can promote the growth of anaerobic bacteria, which produce harmful gases like methane and hydrogen sulfide.
Impact on Invertebrates:
Even invertebrates can be affected by low DO, with some species leaving areas where DO is below certain thresholds, and others experiencing reduced growth and survival.
In summary, low DO levels disrupt the delicate balance of aquatic ecosystems, impacting the health and survival of various organisms.
What is COD in Water?
Chemical Oxygen Demand (COD) in water refers to the amount of oxygen required to chemically oxidize all the organic and inorganic compounds present in a water sample. It's a measure of the total oxygen-demanding potential of the water, indicating the level of organic pollution. Essentially, COD represents the total amount of oxygen needed to break down all the organic matter through chemical oxidation.
What will be happen if COD levels become higher than Acceptable Limit?
If the Chemical Oxygen Demand (COD) level in water becomes higher than normal, it means the water contains more organic matter that can be chemically oxidized, ultimately leading to a reduction in dissolved oxygen (DO) levels. This can harm aquatic life, disrupt ecosystems, and potentially lead to the formation of harmful byproducts during wastewater treatment.
Here's a more detailed explanation:
Oxygen Depletion:
High COD indicates more organic matter, which requires oxygen for breakdown. This increased demand for oxygen can deplete the water's dissolved oxygen, creating a hypoxic or anoxic environment, harmful to aquatic organisms like fish.
Ecosystem Disruption:
Reduced DO levels can stress or kill aquatic organisms, leading to imbalances in the ecosystem and potential loss of biodiversity.
Wastewater Treatment Concerns:
High COD in wastewater can create problems during treatment. Organic compounds can react with chlorine disinfectants, forming trihalomethanes (THMs), some of which are carcinogenic. Additionally, high organic content in effluent can contribute to pollution in receiving waterways and water supplies.
Pollution Indicator:
High COD levels are an indicator of organic pollution in water, reflecting the presence of decaying plant matter, human waste, or industrial effluent.
What will be happen if BOD levels become higher than Acceptable Limit?
If Biochemical Oxygen Demand (BOD) levels are higher than normal, it indicates increased pollution and can lead to several negative consequences for the environment and human health. Specifically, high BOD can cause oxygen depletion in water, harming aquatic life, reducing water quality, and potentially impacting human activities like recreation and water supply.
Here's a more detailed look at what happens when BOD is high:
1. Oxygen Depletion and Aquatic Life:
High BOD signifies a larger amount of organic matter in the water, which bacteria consume, drawing oxygen out of the water. This oxygen depletion can suffocate fish and other aquatic organisms, leading to fish kills and reduced biodiversity.
In severe cases, areas with very low oxygen can become "dead zones" where few or no aquatic life can survive.
2. Water Quality Degradation:
High BOD often coincides with increased levels of other pollutants, such as nutrients, pathogens, and toxic substances. These pollutants can further degrade water quality, making it unsuitable for recreational activities like swimming and fishing. The presence of high BOD can also cause foul odors and discoloration in the water, further reducing its attractiveness.
3. Nutrient Imbalances and Eutrophication:
Excess organic matter from high BOD can disrupt nutrient cycling within the aquatic ecosystem.
This can lead to imbalances in nutrient availability, affecting different organisms in the food web.
High BOD can also contribute to eutrophication, where excessive nutrient enrichment triggers algal blooms. Algal blooms can consume oxygen in the water, further impacting aquatic life and can produce toxins harmful to humans.
4. Impact on Human Activities:
High BOD levels can make water bodies unsuitable for recreational activities like swimming, boating, and fishing. It can also contaminate water sources, posing health risks to humans who consume or use the water.
In essence, high BOD levels indicate a serious problem with water quality, posing threats to aquatic ecosystems, human health, and various human activities that depend on clean water sources.
What happen if BOD levels becomes lower than acceptable limit?
If water's BOD (Biochemical Oxygen Demand) levels are lower than the acceptable limit, it indicates the water is relatively clean with less organic matter and less oxygen demand for microorganisms. Lower BOD levels generally mean better water quality, as there is less organic pollution to deplete dissolved oxygen and harm aquatic life.
Here's a more detailed explanation:
Low BOD = Clean Water:
When BOD is low, it signifies that there's less organic matter present in the water. This means fewer microorganisms are needed to break down the organic matter, reducing the demand for oxygen.
Benefits of Low BOD:
Healthy Aquatic Life: A lower BOD means more dissolved oxygen remains available for aquatic organisms, supporting a healthier ecosystem.
Suitable for Various Uses: Water with low BOD is more suitable for various uses, including drinking, recreation, and irrigation.
Monitoring and Management:
Wastewater treatment plants actively manage BOD levels to ensure water quality is maintained and that regulations are met.
Acceptable Limits:
While acceptable BOD limits vary depending on the intended use of the water, generally, BOD levels below 5 mg/L are considered ideal for drinking water. Treated wastewater discharged into water bodies often has a BOD between 30 and 100 mg/L.
What is Hard Water & Soft water?
Hard water contains dissolved minerals, primarily calcium and magnesium while soft water has had these minerals removed or has low levels of them.
pH of Hard water generally has a higher pH, often above 8.5, making it more alkaline.
Soft water typically has a lower pH, often below 7, making it more acidic.
* How to test Hardness of Potable Water?
* How to test Heavy metal of Potable Water?
* How to test Arsenic in Potable Water?
Potable Water Analysis
Heavy Metal Test
Acceptance Criteria: NMT 0.1 mg/L
Reagent Requirement:
☞ Thioacetamide
☞ 1 N Sodium Hydroxide
☞ 85% Glycerol
☞ Ammonium Acetate
☞ 7 N Hydrochloric Acid
☞ 6 M Ammonium Hydroxide
☞ Lead (II) Nitrate
Reagent Preparation:
☞ Thioacetamide (4%) solution:
Dissolve 4.0 g of thioacetamide in sufficient water to 100 ml.
☞ Mixture for Thioacetamide Reagent: Mix 75 ml of 1N Sodium hydroxide, 25 ml of water and 100 ml of 85% Glycerol together.
☞ Thioacetamide reagent:
Mix 0.2 ml of Thioacetamide (4%) solution and 1ml of mixture for thioacetamide immediately before use and heat on a water bath for 20 second and cool.
☞ Acetate Buffer pH 3.5:
Take 25.0g of Ammonium Acetate to 25 ml of water and add 38 ml of 7N Hydrochloric acid to it and adjust the pH to 3.5 with 6M Ammonium Hydroxide.
☞ Lead Standard Solution (0.1% Pb): Dissolve 0.400g of Lead (II) Nitrate in suuficient water to produce 250 ml.
☞ Lead Standard Solution (100 ppm Pb): Dilute 1 volume of lead standard solution (0.1% Pb) to 10 volumes with water immediately before use.
☞ Lead Standard Solution (10 ppm Pb): Dilute 1 volume of lead standard solution (100 ppm Pb) to 10 volumes with water immediately before use.
☞ Lead Standard Solution (1 ppm Pb): Dilute 1 volume of lead standard solution (10 ppm Pb) to 10 volumes with water immediately before use.
☞ Test solution: Heat 200 ml in a glass evaporating diah on a water bath until the volume is reduced to 20 ml.
Procedure:
12 ml of test solution conplies with the limit test heavy metals.
Sample Preparation: To 12 ml of solution add 2 ml of acetate buffer pH 3.5 and shake, to it add 1.2 ml of thioacetamide reagent mix immediately and allow to stand for 2 minutes.
Standard preparation: To 10 ml of lead standard 1 ppm add 2 ml of the solutiob being tested and volume up to 14 ml with water. Then add 2 ml of acetate pH 3.5 and shake, to it add 1.2 ml of thioacetamide reagent, mix immediately and allow to stand for 2 minutes.
Result: The standard preparation will have faint brown colour which is more than the sample preparation (Maximum 0.1 ppm)
Arsenic Test
Acceptance Criteria: NMT 10 ppb
Required Reagent:
☞ Sodium Hydroxide
☞ Arsenic Trioxide
☞ Potassium Iodide
☞ Stannous Chloride TS
☞ Isopropyl Alcohol
☞ Lead Acetate Solution
☞ Silver Diethyldithiocarbamate TS
☞ Granular Zinc
☞ Hydrochloric Acid
Reagent Preparation:
1. 2N Sulfuric Acid
2. Potassium Iodide TS
3. Silver Diethyldithiocarbamate TS
4. Stannous Chloride TS
5. Arsenic Trioxide Stock solution
6. Standard Arsenic Solution
7. Standard Preparation
8. Test Preparation
Standard Preparation: Pipette 3.0 mL of Standard Arsenic solution into a generator flask and dilute with water to 35 mL.
Test Preparation: Unless otherwise directed in the individual monograph, transfer to the generator flask the quantity in g of the test substance calculated by the formula:
3.0/L in which L is the arsenic limit in ppm, dissolve in water and dilute with water to 35 mL.
Procedure:
Treat the standard preparation and the test preparation as follows:
☞ In both Standard and test preparation, add 20 mL of 7 N Sulfuric acid, 2mL of Potassium Iodide TS, 0.5 mL of Stronger acid stannous chloride TS and 1 ml of Isopropyl alcohol and mix well.
☞ Allow to stand at room temperature for 30 minutes.
☞ Pack the scrubber tube with two pledgets of cotton that have been soaked in saturated lead acetate solution, freed from excess solution be expression, and dried in vacuum at room temperature, leaving a 2 mm space between the two pledgets.
☞ Lubricate the joints (b and d) with suitable stopcock grease designed for use with organic solvents and connect the scrubber unit to the absorber tube (e).
☞ Transfer 3.0 mL of Silver diethyldithiocarbamate TS to the absorber tube.
☞ Add 3.0 g of granular zinc (No. 20 mesh) to the mixture in the flask, immeduately connect the assembled scrubber unit and allow the evolution of hydroven and the color development to proceed at room temperature for 45 minutes, swirling the flask gently at 10-minute intervals.
☞ Disconnect the absorber tube from the generator and scrubber units, and transfer the absorbing solution to a 1-cm absorption cell.
Result: Any red color produced by the Test Preparation does not exceed that produced by the standard preparation. If necessary or desirable, determine the absorbance at the wavelength of maximum absorbance between 535 and 540 nm, with a suitable spectrophotometer or colorimeter, using silver diethyldithiocarbamate TS as the blank.
Total Hardness Tes
Acceptance Criteria: NMT 500 mg/L
Required Reagent:
☞ Sodium Hydroxide
☞ Ammonium Chloride
☞ Ammonia Solution (25%)
☞ Disodium-Magnesium Salt of EDTA
☞ Hydrochloric Acid
☞ Eriochrome Black T
☞ Ethanol
Reagent Preparation
1. Buffer Solution (pH 10 ±0.1)
2. Eriochrome Black T solution 0.5%
3. 0.1M EDTA VS
Determination of Total Hardness of Water:
☞ Pipette 50.0 mL of the sample (Water) into a 250mL conical flask and dilute to 100 mL, preferably with deionized water.
☞ Add 4 mL of buffer solution and 6 drops of the Eriochrome Black T solution.
☞ The colur of the solution should now turn to claret or violet and its pH value should be 10±0.1.
☞ Titrate with the EDTA solution, rather rapidly at the beginning and slowly towards the end of the titration.
☞ Add the EDTA solution until the colour of the solution starts to change from claret or violet to blue and then to a distinct blue endpoint.
CaCO3 Content (mg/L) = (V × E(CaCO3) × 1000)/50
Where,
V = Volume of 0.1M EDTA solution
E = Equivalent weight of CaCO3 (10.01 mg)
TS & VS
* How to prepare 1 M & 1N 250 ml 37% Hcl?
* How to prepare 1 M & 1N 500 ml 98% HcL?
* How to calculate equivalent weight?
* TS vs VS
* Why we have to be calculate factor for Volumetric solution?
* How to Prepare Sodium thiosulfate VS?
* How to prepare Ammonium Iron (II) Sulfate VS?
* How to Prepare EDTA VS?
Primary Standard: A primary standard is a reagent that is extremely pure, stable, has no waters of hydration and has a high molecular weight. In chemistry, a primary standard is a reliable, readily qualified substance.
Reagent: Reagent is any chemical substance required for testing of materialsmproducts.
Test Solutions: Test solutions are solutions of reagents in such solvents and of such define concentrations as to be suitable for specified purpose.
Volumetric solution: Volumetric solutions are solutions of reagents of known concentration intended primarily for use in quantitative determinations.
Volumetric solutions shall not differ from prescribed strength by more thanb10% and the factor shall be determined in triplicate and RSD shall not be more than 0.5%.
Volumetric solutions shall be standaridized by titration against a primary standard or by titration with a standard solution that has been recently standardized against a primary standard.
Buffer solution: A buffer solution is an aquous solution consisting of a mixture of a weak acid and its conjugate base, or vice versa. Its pH changes very little when a small amount of strong acid or base is added to it. Buffer solutions are used as a means of keeping pH at a nearly constant value in a wide variety of chemical applications.
Indicator: Indicators are substances which show a change in color when brought in cotact with acids and bases.
Preparation & Standardization of 0.1M Ammonium Iron (II) Sulfate VS
Preparation: Dissolve 40g of Ammonium iron (II) sulfate in 100mL of 2M sulfuric acid and dilute with sufficient freshly bpiled and cooled water to produce 1000mL.
Standardization: Ascertain its exact concentration in the following manner:
To 25 mL add 10 mL of 1M sulfuric acid and 1mL of orthophosphoric acid and titrate with 0.02M potassium permanganate VS. Each mL of 0.02M potassium permanganate VS is equivalent to 39.21 mg of (NH4)Fe(,SO4)2.6H2O
Preparation & Standardization of 0.1M EDTA VS:
Preparation:
☞ Dissolve 37.5 g of Sodium Edetate in 500 mL of Water.
☞ Add 100 mL of 1 M sodium hydroxide
☞ Dilute up to 1000.0 mL with water.
Standardization:
☞ Dissolve 0.120g of Zinc in 4 mL 7M of Hydrochloric Acid.
☞ Add 2M of Sodium hydroxide solution until the solution is weakly acid and carry out the assay of zinc by complexometry.
☞ 1mL of 0.1M Sodium Edetate is equivalent to 6.538 mg of Zn. Storage in a polyethylene container.
* Importance of Factor determination for volumetric solution
The determination of a factor in volumetric analysis (titration) is crucial because it ensures the accuracy and reliability of analytical results. Here's why it's important:
1. Standardization of Solutions: Many reagents used in titration (e.g., NaOH, KMnO₄) cannot be prepared as exact standard solutions due to their instability or reaction with air. Therefore, they must be standardized against a primary standard to determine their exact concentration — this process gives us the factor.
2. Accurate Concentration Calculation: The factor corrects for any discrepancy between the assumed (nominal) and actual concentration of the titrant, allowing accurate calculation of unknown sample concentrations.
3. Consistency in Results: Using a known factor ensures that results are consistent across different batches, labs, or times, even if the titrant slightly varies in strength.
4. Error Minimization: Without knowing the exact factor, calculations would introduce systematic errors, leading to unreliable or invalid conclusions in chemical analysis.
To calculate the equivalent weight of potassium permanganate (KMnO₄), you need to know:
1. Molecular weight of KMnO₄.
2. Number of electrons transferred in the redox reaction — which depends on the medium (acidic, basic, or neutral).
Step-by-step:
1. Molecular weight of KMnO₄:
K = 39.1
Mn = 54.9
O₄ = 16 × 4 = 64
Total = 39.1 + 54.9 + 64 = 158 g/mol
2. Determine the medium:
In acidic medium, KMnO₄ acts as an oxidizing agent and is reduced from Mn⁷⁺ to Mn²⁺:
MnO₄⁻ → Mn²⁺
Change in oxidation state = 7 − 2 = 5 electrons
3. Equivalent weight formula:
Equivalent weight = Molecular weight\no. of electrons transferred
So in acidic medium:
Equivalent weight of KMnO₄ =158/5}= 31.6 g/equiv
HPLC/UPLC/GC Column Management
Instrument Calibration
* Calibration of FTIR
* Calibration of UV Spectrophotometer
* Calibration of Viscometer
* Calibration of Osmometer
* Calibration of Viscometer
1. Calibration fluids that enable one to check the calibration and linearity of the instrument over its operating torque range of 10%–100% torque.
2. Any spindle and anyone of several calibration fluids may be used to perform a calibration check.
3. Place the viscosity standard fluid in a 600 ml low Griffin beaker into the water bath.
4. The viscosity standard fluid should be immersed in the bath for a minimum of one hour, stirting the fluid perioically and prior to taking measurements.
5. After one hour, check the temperature of the viscosity standard fluid with a thermometer. It must be within ±0.1° of 25°C.
6. Attach the desired spindle to the viscometer.
7. Measure the viscosity using a suitable speed of the three standard fluids and record the readings.
8. Remove the spindle clean and place it proper box.
9. Calculation for the acceptance limit of tha standard fluid:
Full scale viscosity range = TK× SMC × 10000/RPM
Where,
TK = Torque constant
SMC = Spindlr Multiplier Constant
RPM = Rotation per Minute
| Spindle | TK | SMC |
|---|---|---|
| LV1 | 0.09373 | 6.4 |
| LV 2 | 0.09373 | 32 |
| LV 3 | 0.09373 | 128 |
| LV 4 | 0.09373 | 640 |
Calibration frequency: Half yearly
* Calibration of Osmometer
1. When the display reads "Osmometer Ready", press the [NEXT] button unyil [CALIB] appears over the left button. Press it to initiate the calibration procedure.
2. Calibration can be cancelled without changing the existing calibration by pressing the [EXIT] button.
3. Display will briefly read " 50 mOsm Calibration" & then prompt insert a 50 mOsm calibration standard.
4. Follow the prompts on the instrument display. When the instrument completes the test and reports the result, remove the sampler and clean the cooling chamber.
5. Conyinue testing 50 mOsm calibration standards until this calibration point is complete.
6. The calibration program will now briefly read "850 mOsm Calibration" and then prompt insert an 850 mOsm calibration standard.
7. Follow the prompts on the instrument display. Continue testing 850 mOsm calibration standards until this calibration point is complete.
8. If the 2000 mOsm calibration has been turned ON in the Setup menu, tgen the calibration program will prompt to insert 2000 mOsm calibration standard.
9. Follow the prompts on the instrument display and continue testing 2000 mOsm calibration standards unyil this,calibration point is complete.
10. If the 2000 mOsm calibration has been turned OFF, the this additional series of calibration tests will be bypassed by tge calibration program.
11. Upon sucessfully calibration, the instrument will briefly display, "Calibration Complete". Then " Osmometer Ready.
12. Draw the linearity graph with the standard 50, 850 and 2000 mOsm calibration standards. The value of linear regression (r2) should be more than 0.995.
13. Verify the calibration by running a Clinitro™ 290 Reference Solution, repear the testing more two times and find out the relative standard deviations. The accepted value of RSD is not more than 2%.
Calibration frequency: Six months.
* Calibration of Dissolution Tester
* Calibration of TOC machine
* Calibration of pH meter
* Calibration of Conductivity Meter
* Calibration of Glassware
Calibration of FTIR
Calibration procedure (FTIR)
Select Macro
Click the EP validation based on EP 9.6
"Macro Execute" Window will appear
Select Run
After comming dialog box select the measurement and fill the inspected by relative humidity and temperature. As you need complete the comment and selecr desired folder.
Remove the sample from the sample compartment.
Put the polstyrene film over the sample holder after a message tells you.
The report would be automatically printed out anf that will show power spectrum., Resolution, wave length accuracy, Reproducibility of wave lengtg, Reproducibility of absorbance, Pass.
Calibration Procedure (ATR)
Set scan parameter as follows:
Mode: Transmittance
No. of Scans: 20
Resolution: 4 cm-1
Apodization: Happ-Genzel
Scan range: 4000 to 700 cm-1
Detector: Standard
Mirror Speed: 2.8 mm/sec
Aperture: Auto
With the accessory removed from the sample compartment, collect a background spectrum.
Place the accessory in the sample compartment.
Collect transmission spectrum using the same collection parameters as used to collect the background spectrum.
The following are the transmission values the accessory should achieve at least with different prism configuration at 1000 cm-1.
| ZnSe | Ge | Diamond/ZnSe |
|---|---|---|
| 5% | 10% | 5% |
Glassware Calibration
Titration
Types of Titration: mainly 4 types of Titration namely:
1. Acid-base tritration
2. Redox titration
3. Precipitation Titration
4. Complexometric Titration
Redox Titration
Examples of Redox titration:
☞ Titration of potassium permanganate against Oxalic Acid
Types of Redox Titration:
1) Oxidimetry
2) Reduximetry
1) Oxidimetry
Definition: Redox titrations where the titrant is an oxidizing agent.
Examples:
☞ Potassium Permanganate titrations
☞ Potassium dichromate titration
☞ Ceric sulfate titrations
☞ Iodimetric titration
2) Reduximetry
Definition: Redox titrations where the titrant is a reducing agent.
Examples:
☞ Iodometric titration: Sodium thiosulfate+
Iodimetric Titration
These are the titration in which free Iodine (Oxidizing agent) is used. When an analyte that is reducing agent is directly titrated with a standard Iodine solution, the method is called Iodimetry. Starch can be used as indicator in Iodimetric titration.
Iodometric Titration (Reduximetric Titration)
Iodometry is an Indirect Titration. In Iodometric titration, an oxidizing agent allowed to react in neutral medium or in acidic medium with excess of Potassium Iodide (reducing agent) to liberate free Iodine. Free Iodine (Oxidizing agent) is titrated against a standard reducing agent.
Oxidizing agent
An oxidizing agent is a chemical species that undergoes a chemical reaction in which it gains one or more electrons. Examples: I2
Reducing agent
A reducing agent is a substance that is oxidized by donating electrons. (Loss one or more electrons). Exmples: Potassium Iodide
Complexometric titration
Conplexometric titration is a volumetric analysis technique that uses the formation of stable, colored complexes to determine the concentration of metal ions in a solution. It relies on the reaction between a metal ion and a chelating agent like EDTA, to form a complex. The endpoint of the titration is detected visually using an indicator, which changes color when the metal ion is displaced from the indicator complex by the chelating agent.
Chelating agent:
Chelating agent is ligand with multiple donor atoms that can silumtaneously bind to a metal ion, forming a stable complex. EDTA is a common example of chelating agent.
Types of Complexometric Titration:
1) Direct Titration
2) Back Titration or Residual Titration
3) Replacement Titration/Substitution Titration
4) Alkalimetric titration
5) Indirect Titration
What is Osmolarity?
Molarity
Molality
Normality
Gram equivalent weight
Volumetric solution
Test solution
Importance of Factor
Pipetting
☞ If the sample is colorless/slighly colored and clear, slowly drop the lower meniscus of the sample to the mark of pipette and if it is highly colored, then slowly drop the upper meniscus of the sample to the mark of the pipette.
☞For non-viscus samples, allows the pipette to drain for about 15 second or enough time after the liquid has been dispensed.
☞For viscous samples, allows the pipette to drain for bout 45 second or enough time after the liquid has been dispensed.
☞ If the sample is viscoua like syrup/suspension, wash the pipette with the diluents and add the washings to the measured portion.
Differences
Differences between UV and IR
1. UV spectroscopy deals with electronic vibration, while IR spectroscopy deals with molecular vibration.
2. Molecular interaction for UV: Electronic transition, ehilr for IR: Vibrational energy (Stretching, bending)
3. Energy involved in UV: Higer energy (promotes electrons), while in IR: Lower energy (induces molecular vibration)
☞ Types of radiation for UV: Ultraviolet (200-400 nm) and visible (400-800 nm)
☞ Type of radiation for IR: Infrared (4000-400 cm-1)
UV is used for analyze conjugated systems, aromatic compounf, metal conplex
IR is used for analyze functional groups in organic compounds.
Solvents used in UV: Ethanol, Hexane etc (Transparent in UV)
Solvents used in IR: KBr, NaCl; often solid functional group
Information provided for UV: Electronic structure, conjugation
Information provided for IR: Presence of functional group, molecular fingerprint.
Detection: UV measures absorbance VS Wavelength (nm)
IR measures transmittance VS Wavenumber (cm-1)
Accrptance criteria for Re-test
| Test Parameter | Acceptance Criteria |
|---|---|
| Appearance | Comply between two Analyst |
| Idengification by IR/UV/HPLC/TLC/et | Comply between two Analyst |
| Average Weight | Between two Analyst = ±3% |
| Thickness | Between two Analyst = ±0.5 mm |
| Hardness | Between two Analyst = ±3.0 Kp |
| Friability | Between two Analyst = ±0.5% |
| Diameter | Between two Analyst = ±0.5 mm |
| LOD | Between two Analyst = ±0.5% |
| Water Content | Between two Analyst = ±0.5% |
| Disintegration Test | Between two Analyst = ±2 minutes |
| Assay | For HPLC/Potentiometer/Titrimetric/UV spectrophotometer/Ion chromatography: between two analyst = ±2 For AAS/GC: Between teo Analyst = ±5% |
| Content Uniformity | Between two Analyst = ±3% (AV) |
| Dissolution | Between two Analyst = ±5% (Mean) |
| Tapped density | Between two Analyst = ±15% |
| Particle size distribution | Between two Analyst = ±15% |
| Solubility | Conply between two analyst |
| Heavy metals | Comply between two Analyst |
| Residue on Ignition | Between two analyst = ±15% |
| pH | Between two Analyst = ±10% |
| Arsenic | Between two Analyst = ±10% |
| Sulpher dioxide | Between two Analyst = ±10% |
| Sieve Test | Brtween two Analyst = ±10% (for each) |
| Specific optical rotation | Between two Analyst = ±10% |
| Melting Point | Between two Analyst = ±2°C |
| Conductivity | Between two Analyst = ±10% |
| Nitrates | Between two Analyst = ±15% |
| Iron | Between two Analyst = ±15% |
| Free Chlorine | Between two Analyst = ±15% |
| TOC | Comply between Two Analyst |
| Total Hardness (Water | Between two Analyst = ±15% |
| Impurity (Related Substances) | |
| Residual Solvents/Organic Volatile impurities by GC |
Calculation
How many mg/L is equal to 1ppm?
Solution:
One thousandth of a gram is one miligram and 1000 ml in one liter.
So that 1 ppm = 1 mg per liter = 1 mg/L
PPM is derived from the fact that the density of water is taken as 1Kg/L = 1000 g/L = 1000000 mg/L and 1mg/L is 1mg/1000000 mg or one part in one million.
Calculation of ppb
1 ppb = 1 part of a substance per 1,000,000,000 parts of the total.
Mathematical Derivation:
By Mass:
If you're measuring mass/mass (e.g., micrograms of a substance in kilograms of sample):
ppb = mass of solute (µg)\mass of solution (kg)
Since:
1 kg = 1,000,000,000 µg
Then:
1 ppb = 1 ug of substance per 1 kg of solution
By Volume (for gases):
In air or gases, ppb can mean volume/volume:
ppb = (volume of gas of interest\total volume of air)× 10^9
By Mole (mol/mol):
ppb = moles of solute/moles of solution
ppb = (moles of solute\moles of solution)×10^9
Calculation of Strength
S =
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