This document discusses validation and calibration of HPLC systems. It defines validation as establishing that an analytical procedure meets requirements for its intended use through laboratory studies. A validation protocol outlines how validation will be conducted. Equipment validation demonstrates that equipment is suitable for use and comparable to routine equipment. Calibration involves demonstrating that an instrument produces results within specified limits compared to a reference standard. The document outlines parameters to validate like accuracy, precision, specificity, range, robustness and more. It provides details on testing these parameters and accepting calibration of modules like the pump, injector, detector and column heating.
Analytical method validation as per ich and usp shreyas B R
Analytical method validation is a process of documenting/ proving that an analytical method provides analytical data acceptable for the intended use.After the development of an analytical procedure, it is must important to assure that the procedure will consistently produce the intended a precise result with high degree of accuracy. The method should give a specific result that may not be affected by external matters. This creates a requirement to validate the analytical procedures. The validation procedures consists of some characteristics parameters that makes the method acceptable with addition of statistical tools.
The drug or drug combination may not be official in any pharmacopoeias.
A proper analytical procedure for the drug may not be available in the literature due to patent regulations.
Analytical methods may not be available for the drug in the form of a formulation due to the interference caused by the formulation excipients.
Analytical methods for the quantitation of the drug in biological fluids may not be available.
Analytical methods for a drug in combination with other drugs may not be available.
The existing analytical procedures may require expensive reagents and solvents. It may also involve cumbersome extraction and separation procedures and these may not be reliable.
Analytical method validation as per ich and usp shreyas B R
Analytical method validation is a process of documenting/ proving that an analytical method provides analytical data acceptable for the intended use.After the development of an analytical procedure, it is must important to assure that the procedure will consistently produce the intended a precise result with high degree of accuracy. The method should give a specific result that may not be affected by external matters. This creates a requirement to validate the analytical procedures. The validation procedures consists of some characteristics parameters that makes the method acceptable with addition of statistical tools.
The drug or drug combination may not be official in any pharmacopoeias.
A proper analytical procedure for the drug may not be available in the literature due to patent regulations.
Analytical methods may not be available for the drug in the form of a formulation due to the interference caused by the formulation excipients.
Analytical methods for the quantitation of the drug in biological fluids may not be available.
Analytical methods for a drug in combination with other drugs may not be available.
The existing analytical procedures may require expensive reagents and solvents. It may also involve cumbersome extraction and separation procedures and these may not be reliable.
The objective of any chemical analytical measurement is to get consistent, reliable and accurate data.
Proper functioning and performance of analytical instruments and computer systems plays a major role in achieving this goal.
Therefore, analytical instrument qualification (AIQ) and calibration should be part of any good analytical practice.
Ion pair chromatography for pharmacy studentsabhishek rai
Ion-PairChromatography
A GENERALISED OVERVIEW
Chromatography
HPLC
Reverse Phase Chromatography
Ion Pair Chromatography
Ion Pair Reagent
Mechanism of Ion Pair Chromatography
Ion Pair Wash Procedure
In this slide contains Introduction, levels of cleaning, mechanism, sampling method of cleaning validation.
Presented by: P. VENKATESH (Department of pharmaceutical analysis).RIPER, anantapur
The versatile instrument is used to isolate unknown compounds from a HPTLC/TLC plate and transfer them into a mass spectrometer for identification or structure elucidation.
The objective of any chemical analytical measurement is to get consistent, reliable and accurate data.
Proper functioning and performance of analytical instruments and computer systems plays a major role in achieving this goal.
Therefore, analytical instrument qualification (AIQ) and calibration should be part of any good analytical practice.
Ion pair chromatography for pharmacy studentsabhishek rai
Ion-PairChromatography
A GENERALISED OVERVIEW
Chromatography
HPLC
Reverse Phase Chromatography
Ion Pair Chromatography
Ion Pair Reagent
Mechanism of Ion Pair Chromatography
Ion Pair Wash Procedure
In this slide contains Introduction, levels of cleaning, mechanism, sampling method of cleaning validation.
Presented by: P. VENKATESH (Department of pharmaceutical analysis).RIPER, anantapur
The versatile instrument is used to isolate unknown compounds from a HPTLC/TLC plate and transfer them into a mass spectrometer for identification or structure elucidation.
The analyst is required to analyze a number of QC samples throughout the run where there are decisions to be made based on a window of acceptance for each QC sample analyzed.
This content is suitable for medical technologists/technicians/lab assistants/scientists writing the SMLTSA board exam. The content is also suitable for biomedical technology students and people also interested in learning about test methodologies used in medical technology. This chapter describes test quality assurance (QA) and quality control (QC). Please note that these notes are a collection I used to study for my board exam and train others who got distinctions using these.
Disclaimer: Credit goes to those who wrote the notes and the examiners of each exam question. Please use only as a reference guide and use your prescribed textbook for the latest and most accurate notes and ranges. The material here is not referenced as it is a collection of pieces of study notes from multiple people, and thus will not be held viable for any misinterpretations. Please use at your own discretion.
This presentation was made to solely for students to make them aware/ understand basics of “Analytical Method Validation”. These slides are part of lectures delivered in M. Pharmacy Curriculum & taken up from various books and websites
Analytical method development and validation are one of the very imp aspects in Drug testing and approval process.Here I tried to explain the same with my experience.
New Directions in Targeted Therapeutic Approaches for Older Adults With Mantl...i3 Health
i3 Health is pleased to make the speaker slides from this activity available for use as a non-accredited self-study or teaching resource.
This slide deck presented by Dr. Kami Maddocks, Professor-Clinical in the Division of Hematology and
Associate Division Director for Ambulatory Operations
The Ohio State University Comprehensive Cancer Center, will provide insight into new directions in targeted therapeutic approaches for older adults with mantle cell lymphoma.
STATEMENT OF NEED
Mantle cell lymphoma (MCL) is a rare, aggressive B-cell non-Hodgkin lymphoma (NHL) accounting for 5% to 7% of all lymphomas. Its prognosis ranges from indolent disease that does not require treatment for years to very aggressive disease, which is associated with poor survival (Silkenstedt et al, 2021). Typically, MCL is diagnosed at advanced stage and in older patients who cannot tolerate intensive therapy (NCCN, 2022). Although recent advances have slightly increased remission rates, recurrence and relapse remain very common, leading to a median overall survival between 3 and 6 years (LLS, 2021). Though there are several effective options, progress is still needed towards establishing an accepted frontline approach for MCL (Castellino et al, 2022). Treatment selection and management of MCL are complicated by the heterogeneity of prognosis, advanced age and comorbidities of patients, and lack of an established standard approach for treatment, making it vital that clinicians be familiar with the latest research and advances in this area. In this activity chaired by Michael Wang, MD, Professor in the Department of Lymphoma & Myeloma at MD Anderson Cancer Center, expert faculty will discuss prognostic factors informing treatment, the promising results of recent trials in new therapeutic approaches, and the implications of treatment resistance in therapeutic selection for MCL.
Target Audience
Hematology/oncology fellows, attending faculty, and other health care professionals involved in the treatment of patients with mantle cell lymphoma (MCL).
Learning Objectives
1.) Identify clinical and biological prognostic factors that can guide treatment decision making for older adults with MCL
2.) Evaluate emerging data on targeted therapeutic approaches for treatment-naive and relapsed/refractory MCL and their applicability to older adults
3.) Assess mechanisms of resistance to targeted therapies for MCL and their implications for treatment selection
ARTIFICIAL INTELLIGENCE IN HEALTHCARE.pdfAnujkumaranit
Artificial intelligence (AI) refers to the simulation of human intelligence processes by machines, especially computer systems. It encompasses tasks such as learning, reasoning, problem-solving, perception, and language understanding. AI technologies are revolutionizing various fields, from healthcare to finance, by enabling machines to perform tasks that typically require human intelligence.
NVBDCP.pptx Nation vector borne disease control programSapna Thakur
NVBDCP was launched in 2003-2004 . Vector-Borne Disease: Disease that results from an infection transmitted to humans and other animals by blood-feeding arthropods, such as mosquitoes, ticks, and fleas. Examples of vector-borne diseases include Dengue fever, West Nile Virus, Lyme disease, and malaria.
Prix Galien International 2024 Forum ProgramLevi Shapiro
June 20, 2024, Prix Galien International and Jerusalem Ethics Forum in ROME. Detailed agenda including panels:
- ADVANCES IN CARDIOLOGY: A NEW PARADIGM IS COMING
- WOMEN’S HEALTH: FERTILITY PRESERVATION
- WHAT’S NEW IN THE TREATMENT OF INFECTIOUS,
ONCOLOGICAL AND INFLAMMATORY SKIN DISEASES?
- ARTIFICIAL INTELLIGENCE AND ETHICS
- GENE THERAPY
- BEYOND BORDERS: GLOBAL INITIATIVES FOR DEMOCRATIZING LIFE SCIENCE TECHNOLOGIES AND PROMOTING ACCESS TO HEALTHCARE
- ETHICAL CHALLENGES IN LIFE SCIENCES
- Prix Galien International Awards Ceremony
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These simplified slides by Dr. Sidra Arshad present an overview of the non-respiratory functions of the respiratory tract.
Learning objectives:
1. Enlist the non-respiratory functions of the respiratory tract
2. Briefly explain how these functions are carried out
3. Discuss the significance of dead space
4. Differentiate between minute ventilation and alveolar ventilation
5. Describe the cough and sneeze reflexes
Study Resources:
1. Chapter 39, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 34, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 17, Human Physiology by Lauralee Sherwood, 9th edition
4. Non-respiratory functions of the lungs https://academic.oup.com/bjaed/article/13/3/98/278874
Couples presenting to the infertility clinic- Do they really have infertility...Sujoy Dasgupta
Dr Sujoy Dasgupta presented the study on "Couples presenting to the infertility clinic- Do they really have infertility? – The unexplored stories of non-consummation" in the 13th Congress of the Asia Pacific Initiative on Reproduction (ASPIRE 2024) at Manila on 24 May, 2024.
Title: Sense of Smell
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
Report Back from SGO 2024: What’s the Latest in Cervical Cancer?bkling
Are you curious about what’s new in cervical cancer research or unsure what the findings mean? Join Dr. Emily Ko, a gynecologic oncologist at Penn Medicine, to learn about the latest updates from the Society of Gynecologic Oncology (SGO) 2024 Annual Meeting on Women’s Cancer. Dr. Ko will discuss what the research presented at the conference means for you and answer your questions about the new developments.
Pulmonary Thromboembolism - etilogy, types, medical- Surgical and nursing man...VarunMahajani
Disruption of blood supply to lung alveoli due to blockage of one or more pulmonary blood vessels is called as Pulmonary thromboembolism. In this presentation we will discuss its causes, types and its management in depth.
Tom Selleck Health: A Comprehensive Look at the Iconic Actor’s Wellness Journeygreendigital
Tom Selleck, an enduring figure in Hollywood. has captivated audiences for decades with his rugged charm, iconic moustache. and memorable roles in television and film. From his breakout role as Thomas Magnum in Magnum P.I. to his current portrayal of Frank Reagan in Blue Bloods. Selleck's career has spanned over 50 years. But beyond his professional achievements. fans have often been curious about Tom Selleck Health. especially as he has aged in the public eye.
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Introduction
Many have been interested in Tom Selleck health. not only because of his enduring presence on screen but also because of the challenges. and lifestyle choices he has faced and made over the years. This article delves into the various aspects of Tom Selleck health. exploring his fitness regimen, diet, mental health. and the challenges he has encountered as he ages. We'll look at how he maintains his well-being. the health issues he has faced, and his approach to ageing .
Early Life and Career
Childhood and Athletic Beginnings
Tom Selleck was born on January 29, 1945, in Detroit, Michigan, and grew up in Sherman Oaks, California. From an early age, he was involved in sports, particularly basketball. which played a significant role in his physical development. His athletic pursuits continued into college. where he attended the University of Southern California (USC) on a basketball scholarship. This early involvement in sports laid a strong foundation for his physical health and disciplined lifestyle.
Transition to Acting
Selleck's transition from an athlete to an actor came with its physical demands. His first significant role in "Magnum P.I." required him to perform various stunts and maintain a fit appearance. This role, which he played from 1980 to 1988. necessitated a rigorous fitness routine to meet the show's demands. setting the stage for his long-term commitment to health and wellness.
Fitness Regimen
Workout Routine
Tom Selleck health and fitness regimen has evolved. adapting to his changing roles and age. During his "Magnum, P.I." days. Selleck's workouts were intense and focused on building and maintaining muscle mass. His routine included weightlifting, cardiovascular exercises. and specific training for the stunts he performed on the show.
Selleck adjusted his fitness routine as he aged to suit his body's needs. Today, his workouts focus on maintaining flexibility, strength, and cardiovascular health. He incorporates low-impact exercises such as swimming, walking, and light weightlifting. This balanced approach helps him stay fit without putting undue strain on his joints and muscles.
Importance of Flexibility and Mobility
In recent years, Selleck has emphasized the importance of flexibility and mobility in his fitness regimen. Understanding the natural decline in muscle mass and joint flexibility with age. he includes stretching and yoga in his routine. These practices help prevent injuries, improve posture, and maintain mobilit
2. Validation, USP:
“Validation of an analytical procedure is the process by which it
is established, by laboratory studies, that the performance
characteristics of the procedure meet the requirements for the
intended analytical applications.”
Validation Protocol:
A written plan stating how validation will be conducted and
defining acceptance criteria. For example, the protocol for a
manufacturing process identifies processing equipment,
critical process parameters/operating ranges, product
characteristics, sampling, test data to be collected, number of
validation runs, and acceptable test results.
Equipment Validation:
Demonstrate that equipment used in validation studies is suitable
for use and is comparable to equipment used for routine analysis. 2
3. •Qualification: Action of proving and documenting that
equipment or ancillary systems are properly installed, work
correctly, and actually lead to the expected results.
•Qualification is part of validation, but the individual qualification
steps alone do not constitute process validation.
Parts Of The Qualification:
The entire process typically consists of four parts:
•Design qualification (DQ),
•Installation qualification(IQ),
•Operational qualification (OQ), and
•Performance qualification (PQ).
3
4. A schematic diagram illustrating the timeline and documents in the
various stages in HPLC system qualification.
4
7. Accuracy:
Definition: The accuracy of an analytical procedure expresses the closeness of
agreement between the value that is accepted either as a conventional true
value or as an accepted reference value and the value found.
How To Determine??
• Known amounts of Related substances and the drug substance in placebo are
spiked to prepare an accuracy sample of known concentration of Related
substance.
• According to the ICH, accuracy should be determined using a minimum of
nine determinations over a minimum of three concentration levels covering
the range(from 50% of the ICH reporting level to 150% of the proposed shelf
life specification of the related substances) specified.
e.g. Level Accuracy
125% 99.6 +/- 0.2%
75% 100.3 +/- 0.8%
25% 99.2 +/- 0.7%
7
(Reference: IJPSR
Determination of Voglibose by HPLC and validation of method)
8. •Intrinsic Accuracy:
Intrinsic accuracy indicates the bias caused by sample matrix and sample
preparation.
•Overall Accuracy:
oMatrix effect
o Sample preparation
o Calculation error
%relative substance(calculated)
•Overall accuracy = -----------------------------------------
%relative substance (theory)
8
9. Precision :
Definition : The Precision is a measure of the ability of the method to generate
reproducible results. The precision of a method is evaluated for repeatability,
intermediate precision, and reproducibility.
•Repeatability is a measure of the ability of the method to generate similar
results for multiple preparations of the same homogeneous sample by one
analyst using the same instrument in a short time duration (e.g., on the same
day). For instance, method repeatability for pharmaceutical assays may be
measured by making six sample determinations at 100% concentration, or by
preparing three samples at 80, 100, and 120% concentration levels each.
•Intermediate precision is a measure of the variability of method results where
samples are tested and compared using different analysts, different equipment,
and on different days, etc. This study is a measure of the intra-laboratory
variability and is a measure of the precision that can be expected within a
laboratory.
•Reproducibility is the precision obtained when samples are prepared and
compared between different testing sites. Method reproducibility is often
assessed during collaborative studies at the time of technology or method
transfer. %RSD= (s/µ)*100 9
10. Limit of Detection (LOD, DL)
– The LOD of an analytical procedure is the lowest amount
of analyte in sample which can be detected but not
necessarily quantitated as an exact value.
• Determination is usually based on
– Signal to noise ratio (~3:1) (baseline noise)
or
– Standard deviation of response (s) and Slope (S)
• 3.3 s/S
– SNR = H/h
Where H = height of the peak corresponding to the component
h = absolute value of the largest noise fluctuation from
the baseline of the chromatogram of a blank
solution
10
11. Limit of Quantitation (LOQ, QL)
– The LOQ is the lowest amount of analyte in a sample
which can be quantitatively determined with suitable
precision and accuracy
– The quantitation limit is used particularly for the determination of
impurities and/or degradation products
• Determination is usually based on
– Signal to noise ratio (~10:1) (baseline noise)
or
– Standard deviation of response (s) and Slope (S)
• 10 s/S
- The Quantitation limit of a method is affected by both the detector
sensitivity and the accuracy of sample preparation.
11
13. Range:
ICH Definition: The range of an analytical procedure is the interval
between the upper and lower concentrations of analytes in the for
which it has been demonstrated that the analytical procedure has a
suitable level of precision, accuracy, and linearity.
Range For Different Tests:
• Assay
80 to 120% of test concentration
• Content uniformity
70 to 130% of test concentration
• Dissolution
Q-20% to 120%
• Impurities
Reporting level – 120% of specification limit (with respect to test
concentration of API)
• Assay & Impurities
Reporting level to 120% of assay specification
13
14. Linearity:
Definition : Linearity of an analytical procedure is its ability
(within a given range) to obtain test results which are
directly proportional to the concentration of analytes in the
sample.
•If there is a linear relationship test results should be
evaluated by appropriate statistical methods like,
•Correlation coefficient
•Y-intercept
•Slope of regression line
•Plot of the Data
•Method To Determine Linearity
•Direct Weigh Method
•Dilution Method
14
16. SYSTEM SUITABILITY TESTING (SST)
• System suitability testing (SST) is used to verify resolution,
column efficiency, and repeatability of the analysis system to
ensure its adequacy for performing the intended application
on a daily basis.
Which Parameters??
•Number of theoretical plates (efficiency)
•Capacity factor,
•Separation (relative retention)
•Resolution,
•Tailing factor
•Relative Standard Deviation (Precision)
16
17. •Plate number or number of theoretical plates (n)
n=L/H, where L is Length of Column
H is HETP or height of one theoretical plate
•Capacity factor (capacity ratio) k
k’= tr-tm/tm where tr is retention time
tm is dead time
•Separation Factor (relative retention)
α=k1/k2 where k1 is capacity factor of compound a
and k2 is capacity factor of compound b
•Tailing factor ,T
T=W/2f where W is width at 5% at peak height
f, distance between max and leading edge of the peak
17
19. Robustness :
Definition : Robustness is reliability of an analytical procedure with
respect to deliberate variations in method parameters.
•If measurements are susceptible to variations in analytical conditions
the analytical conditions should be suitably controlled or a
precautionary statement should be included in the procedure.
•PARAMETERS TO BE EVALUATED FOR ROBUSTNESS
•Column consistency
•Three columns packed by bonded phases from three different
silica lots
•Mobile Phase
• pH (±0.1–0.2 units)
• Buffer concentration (±5–10mM)
• Percentage organic modifier (±1–2% MP)
•Sample
•Injection volume or sample concentration
• Solvent strength for the final solution
•Column temperature (±5°C)
•Detector wavelength (±3nm) 19
20. Selectivity and Specificity :
•Selectivity is the ability to measure accurately and specifically the
analyte in the presence of components that may be expected to be
present in the sample matrix.
•Specificity for an assay ensures that the signal measured comes from
the substance of interest, and that there is no interference from
excipient s and/or degradation products and/or impurities.
20
21. Calibration:
Definition (ICH): The demonstration that a particular
instrument or device produces results within specified limits
by comparison with those produced by a reference or
traceable standard over an appropriate range of
measurements.
Various Calibration parameters for HPLC are:
•Flow rate accuracy (Pump Module)
• Injector accuracy
•Carryover
•System Precision
•Wavelength accuracy
•Detector linearity
• Injector linearity
• Gradient Performance Check
• Column oven temperature accuracy
21
22. Flow Rate Accuracy:
1. Prime all the solvent lines with water.
2. Set the flow rate to 0.500 ml/m.
3.Wait for about 15 m to stabilize the system and ensure that the pressure is stable.
4.Insert the outlet tubing into a 10 ml volumetric flask and start the stop watch
simultaneously.
5. Stop the stopwatch when the lower meniscus reaches the 10 ml mark on the
flask.
6. Record the elapsed time.
7. Similarly check the flow for 1.0 ml/m and 2.0 ml/m.
Acceptance criteria:
The time taken to collect the water should be with in ± 2.0% of the actual value.
22
23. Pressure Test :
•The performance of the HPLC pump depends on the proper functioning
of the check valves and the proper connection of the tubing.
•For pump systems that output the pressure reading in the pump head
over time, a simple pressure test can be a useful qualitative test to check
the condition of the check valves and to determine whether or not there
are any leaks in the system.
•The first step of the pressure test is to plug the outlet of the pump using
a dead-nut and by setting the automatic pump shutdown pressure to
6,000 psi.
•The pump-head pressure signal output is connected to a recorder.
Pressurize the pump by pumping methanol at 1 mL/min. The pressure
inside the pump head increases quickly as the outlet of the pump is
blocked. As the pressure increases to about 3,000 psi, the flow rate is
reduced to 0.1 mL/min.
•The pressure will gradually rise to the shutdown pressure if the check
valves are able to hold the mobile phase in the pump chamber as would
be normally expected . If the check valve is not functioning properly, the
pressure will fluctuate at about 3,000 psi instead of reaching the
shutdown pressure.
23
25. Injector Accuracy:
1. Connect the pump and detector inlet with union.
2. Prepare mobile phase consisting of a mixture of water and Methanol (70:30)
3. Set a flow rate of 0.5 ml/m and a run time of 1 m.
4. Set the column temperature at 25± 2°C.
5.Fill a standard HPLC vial to 2/3rd with water. Seal the vial properly with a
cap.
6. Weigh the vial and record the weight as W1 grams.
7. Place the vial in the chromatographic system and perform 6 injections of
50μl volume from this vial.
8. Weigh the vial again and note the weigh after the injections as W2 grams.
9. Calculate the mean volume injected per injection as follows:
•Mean injected volume (μl) = (W1 – W2) × 100/6
•Acceptance criteria: The mean injected volume should be 50.0±1.0 μl.
25
Reference:
•JBSR (Journal)
•Analytical Method Validation and Instrument Performance Verification
-Herman Lam and Y.C. Lec
26. Carryover :
•Small amounts of analyte may get carried over from the previous
injection and contaminate the next sample to be injected.
•The carryover will affect the accurate quantitation of the subsequent
sample.
•The problem is more serious when a dilute sample is injected after a
concentrated sample.
•In order to avoid cross contamination from the previous sample
injection, all the parts in the injector that come into contact with the
sample (the injection loop, the injection needle and the needle seat)
have to be cleaned effectively after the injection.
•The effectiveness of the cleaning can be evaluated by injecting a blank
after a sample that contains a high concentration of analyte.
•The response of the analyte found in the blank sample expressed as a
percentage of the response of the concentrated sample can be used to
determine the level of carryover. 26
27. Detector Module:
(A)UV-Visible Detector Module:
•Wavelength Accuracy
1. Wavelength accuracy is defined as the deviation of the
wavelength reading at an absorption or emission band from the
known wavelength of the band.
2. The deuterium line at 656 nm and the absorption bands at 418,
453 and 536 nm in a Holmium oxide filter are often used.
(B) PDA Detector Accuracy:
•Select 3D mode and set the wavelength range as 200-400nm.
•Inject 20 μl Holonium oxide of standard preparation once into the
chromatographic system.
•Extract and record the chromatograms at wavelengths of 202 to 208nm
with an interval of 1nm and at 269 to 275 nm with an interval of 1nm.
•Note down the height or absorbance.
Acceptance criteria: The maximum absorbance should be at 205±2nm
and 272±2nm.
27
28. 28
You can Calibrate the PDA Detector for 5 Parameters:
1-Baseline Noise and PDA Drift.
2- Your Lamp Intensity.
3- You have to check for the Wavelength Accuracy.
• You can inject Pyrene with Methanol as eluent at a flow of 1 ml/min.
• The characteristically maximum of pyrene is determined at 333 nm and
compared to a theoretical value.
4- Lastly- You must check your PDA's Linearity
you can inject 5 caffeine solutions at different concentrations.
Concentrations and peak heights can be represented in a graph. The
regression coefficient of the resulting line and the deviations from it
indicate indicates the linearity- Usually you R square value should be
above 99.90% .
32. Noise and Drift :
•Drift: upward &/or downward movement of base line.
•Noise : any unwanted signal recorded by the detector.
•Electronic, pump and photometric noise, poor lamp intensity,
dirty flow cell, and thermal instability contribute to the overall
noise and drift in the detector.
•Excessive noise can reduce the sensitivity of the detector and
hence affect the quantitation of low-level analytes.
•Detector drift may affect the baseline determination and peak
integration.
•Overcomes for avoiding Noise and Drift:
•The detector should be warmed up prior to the test, and any
temperature fluctuations should be avoided during the test.
•For a dynamic testing condition, methanol is passed through the
flow cell at 1 mL/min.
•A backpressure of about 500 psi is maintained to prevent bubble
formation.
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33. Column Heating Module :
•The efficiency of a HPLC column varies with column temperature.
•capacity factor k’ decreases with temperature, and hence the
retention of the analysis decreases with temperature .
•Retention drops by 1 to 3% for each increase of 1◦C.
How To Check??
•The temperature accuracy of the column heater is evaluated by
placing a calibrated thermometer in the column compartment to
measure the actual compartment temperature.
•The thermometer readings are compared to the preset
temperature at 40 ◦C and 60◦C.
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