Types of process validation
Depending on when it is performed in relation to production, validation can be prospective, concurrent, retrospective or revalidation (repeated validation).
Prospective validation
Itis carried out during the development stage by means of a risk analysis of the production process, which is broken down into individual steps: these are then evaluated on the basis of past experience to determine whether they might lead to critical situations.
Where possible critical situations are identified, the risk is evaluated, the potential causes are investigated and assessed for probability and extent, the trial plans are drawn up, and the priorities set. The trials are then performed and evaluated, and an overall assessment is made. If, at the end, the results are acceptable, the process is satisfactory. Unsatisfactory processes must be modified and improved until a validation exercise proves them to be satisfactory. This form of validation is essential in order to limit the risk of errors occurring on the production scale, e.g. in the preparation of injectable products.
Concurrent validation
is carried out during normal production. This method is effective only if the development stage has resulted in a proper understanding of the fundamentals of the process. The first three production-scale batches must be monitored as comprehensively as possible.1The nature and specifications of subsequent in-process and final tests are based on the evaluation of the results of such monitoring.
1 This careful monitoring of the first three production batches is sometimes regarded as prospective validation.
Concurrent validation together with a trend analysis including stability should be carried out to an appropriate extent throughout the life of the product.
Retrospective validation
involves the examination of past experience of production on the assumption that composition, procedures, and equipment remain unchanged; such experience and the results of in-process and final control tests are then evaluated. Recorded difficulties and failures in production are analysed to determine the limits of process parameters. A trend analysis may be conducted to determine the extent to which the process parameters are within the permissible range.
Retrospective validation is obviously not a quality assurance measure in itself, and should never be applied to new processes or products. It may be considered in special circumstances only, e.g. when validation requirements are first introduced in a company. Retrospective validation may then be useful in establishing the priorities for the validation programme. If the results of a retrospective validation are positive, this indicates that the process is not in need of immediate attention and may be validated in accordance with the normal schedule. For tablets which have been compressed under individual pressure-sensitive cells, and with qualified equipment, retrospective validation is the most comprehensive test of the overall manufacturing process of this dosage form. On the other hand, it should not be applied in the manufacture of sterile products.
Revalidation
is needed to ensure that changes in the process and/or in the process environment, whether intentional or unintentional, do not adversely affect process characteristics and product quality.
Revalidation may be divided into two broad categories:
• Revalidation after any change having a bearing on product quality.
• Periodic revalidation carried out at scheduled intervals.
Revalidation after changes. Revalidation must be performed on introduction of any changes affecting a manufacturing and/or standard procedure having a bearing on the established product performance characteristics. Such changes may include those in starting material, packaging material, manufacturing processes, equipment, in-process controls, manufacturing areas, or support systems (water, steam, etc.). Every such change requested should be reviewed by a qualified validation group, which will decide whether it is significant enough to justify revalidation and, if so, its extent.
Revalidation after changes may be based on the performance of the same tests and activities as those used during the original validation, including tests on subprocesses and on the equipment concerned. Some typical changes which require revalidation include the following:
• Changes in the starting material(s). Changes in the physical properties, such as density, viscosity, particle size distribution, and crystal type and modification, of the active ingredients or excipients may affect the mechanical properties of the material; as a consequence, they may adversely affect the process or the product.
• Changes in the packaging material, e.g. replacing plastics by glass, may require changes in the packaging procedure and therefore affect product stability.
• Changes in the process, e.g. changes in mixing time, drying temperature and cooling regime, may affect subsequent process steps and product quality.
• Changes in equipment, including measuring instruments, may affect both the process and the product; repair and maintenance work, such as the replacement of major equipment components, may affect the process.
• Changes in the production area and support system, e.g. the rearrangement of manufacturing areas and/or support systems, may result in changes in the process. The repair and maintenance of support systems, such as ventilation, may change the environmental conditions and, as a consequence, revalidation/requalification may be necessary, mainly in the manufacture of sterile products.
• Unexpected changes and deviations may be observed during self-inspection or audit, or during the continuous trend analysis of process data.
Periodic revalidation. It is well known that process changes may occur gradually even if experienced operators work correctly according to established methods. Similarly, equipment wear may also cause gradual changes. Consequently, revalidation at scheduled times is advisable even if no changes have been deliberately made.
The decision to introduce periodic revalidation should be based essentially on a review of historical data, i.e. data generated during in-process and finished product testing after the latest validation, aimed at verifying that the process is under control. During the review of such historical data, any trend in the data collected should be evaluated.
In some processes, such as sterilization, additional process testing is required to complement the historical data. The degree of testing required will be apparent from the original validation.
Additionally, the following points should be checked at the time of a scheduled revalidation:
• Have any changes in master formula and methods, batch size, etc., occurred? If so, has their impact on the product been assessed?
• Have calibrations been made in accordance with the established programme and time schedule?
• Has preventive maintenance been performed in accordance with the programme and time schedule?
• Have the standard operating procedures (SOPs) been properly updated?
• Have the SOPs been implemented?
• Have the cleaning and hygiene programmes been carried out?
• Have any changes been made in the analytical control methods?
Sunday, February 1, 2009
Electrophoresis
Electrophoresis
Principles of Gel Electrophoresis
Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge or conformation. As such, it is one of the most widely-used techniques in biochemistry and molecular biology.
When charged molecules are placed in an electric field, they migrate toward either the positive or negative pole according to their charge. In contrast to proteins, which can have either a net positive or net negative charge, nucleic acids have a consistent negative charge imparted by their phosphate backbone, and migrate toward the anode.
Proteins and nucleic acids are electrophoresed within a matrix or "gel". Most commonly, the gel is cast in the shape of a thin slab, with wells for loading the sample. The gel is immersed within an electrophoresis buffer that provides ions to carry a current and some type of buffer to maintain the pH at a relatively constant value.
The gel itself is composed of either agarose or polyacrylamide, each of which have attributes suitable to particular tasks:
Agarose is a polysaccharide extracted from seaweed. It is typically used at concentrations of 0.5 to 2%. The higher the agarose concentration the "stiffer" the gel. Agarose gels are extremely easy to prepare: you simply mix agarose powder with buffer solution, melt it by heating, and pour the gel. It is also non-toxic.
Agarose gels have a large range of separation, but relatively low resolving power. By varying the concentration of agarose, fragments of DNA from about 200 to 50,000 bp can be separated using standard electrophoretic techniques.
Polyacrylamide is a cross-linked polymer of acrylamide. The length of the polymer chains is dictated by the concentration of acrylamide used, which is typically between 3.5 and 20%.
Polyacrylamide gels are significantly more annoying to prepare than agarose gels. Because oxygen inhibits the polymerization process, they must be poured between glass plates (or cylinders).
Acrylamide is a potent neurotoxin and should be handled with care! Wear disposable gloves when handling solutions of acrylamide, and a mask when weighing out powder. Polyacrylamide is considered to be non-toxic, but polyacrylamide gels should also be handled with gloves due to the possible presence of free acrylamide.
Polyacrylamide gels have a rather small range of separation, but very high resolving power. In the case of DNA, polyacrylamide is used for separating fragments of less than about 500 bp. However, under appropriate conditions, fragments of DNA differing is length by a single base pair are easily resolved. In contrast to agarose, polyacrylamide gels are used extensively for separating and characterizing mixtures of proteins.
Cross section diagram of gel apparatus design:
A. Tube
B. Vertical Slab
C. Horizontal submarine
D. Horizontal thin layer with buffer pads
Principle of Separation
• According to charge
• According to size
(1) According to charge
• When charged molecules are placed in an electric field, they migrate toward either the positive (anode) or negative (cathode) pole according to their charge.
Instruments and Reagents
• Buffer
• Power supply
• Supporting media
• Detection and Quantification
I. Buffer
• Function of buffer
• 1. carries the applied current
• 2. established the pH
• 3. determine the electric charge on the solute
• High ionic strength of buffer
• produce sharper band
• produce more heat
• Commonly used buffer
• Barbital buffer & Tris-EDTA for protein
• Tris-acetate-EDTA & Tris-borate-EDTA (50mmol/L; pH 7.5-7.8)
II. Power Supply
• Function: To supply electric power
• Constant voltage (increasing of heat over time
• Heat = E x I x t
• Constant current
• according to Ohm’s law E= I x R
• R decrease over time, which inturn decrease the heat effect
E= EMF in volts (V)
I = Current in amperes (A)
t = Time in second (s)
III.Support materials used in electrophoresis
• Paper
• Starch
• Agar/agarose
• Cellulose acetate
• polyacrylamide gel
Support Media (ii)
• Agarose and polyacrylamide gels are across-linked, spongelike structure
• It is important that the support media is electrically neutral. Presence of charge group may cause:
• -Migration retardation
-The flow of water toward one or the other electrode so called ‘Electroendosmosis (EEO)’, which decrease resolution of the separation
(1) Agarose
• What is agarose?
• a chain of sugar molecules
• Extracted from seaweed
(1) Agarose gels (ii)
• For the separation of (1) large protein or protein complex (2) polynucleotide 50-30,000 base-pairs
The pore size is determined by adjusting the concentration of agarose in a gel (normally in the rank of 0.4-4% OH, O, CH2OH,
(2) Polyacrylamide gels
CH2=CHCONH2 + CH2(NHCOHC=CH2)2
Acrylamide N,N,N,N-methylenebisacrylamide
Free radical catalyst
-CH2-CH-CH2-CH-CH2-CH-
-CH2-CH-CH2-CH-CH2-CH-
CO, NH, CH2
A. Agarose gel B. Polyacrylamide gel
Polymerization of acrylamide gel can be initiated either by a chemical peroxide or by photochemical method
1. The most common method, ammonium persulphate as the initiator peroxide and TEMED as the catalyst
2. For photochemical polymerization, riboflavin and long-wave UV light are initiator and TEMED as the catalyst
Principles of Gel Electrophoresis
Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge or conformation. As such, it is one of the most widely-used techniques in biochemistry and molecular biology.
When charged molecules are placed in an electric field, they migrate toward either the positive or negative pole according to their charge. In contrast to proteins, which can have either a net positive or net negative charge, nucleic acids have a consistent negative charge imparted by their phosphate backbone, and migrate toward the anode.
Proteins and nucleic acids are electrophoresed within a matrix or "gel". Most commonly, the gel is cast in the shape of a thin slab, with wells for loading the sample. The gel is immersed within an electrophoresis buffer that provides ions to carry a current and some type of buffer to maintain the pH at a relatively constant value.
The gel itself is composed of either agarose or polyacrylamide, each of which have attributes suitable to particular tasks:
Agarose is a polysaccharide extracted from seaweed. It is typically used at concentrations of 0.5 to 2%. The higher the agarose concentration the "stiffer" the gel. Agarose gels are extremely easy to prepare: you simply mix agarose powder with buffer solution, melt it by heating, and pour the gel. It is also non-toxic.
Agarose gels have a large range of separation, but relatively low resolving power. By varying the concentration of agarose, fragments of DNA from about 200 to 50,000 bp can be separated using standard electrophoretic techniques.
Polyacrylamide is a cross-linked polymer of acrylamide. The length of the polymer chains is dictated by the concentration of acrylamide used, which is typically between 3.5 and 20%.
Polyacrylamide gels are significantly more annoying to prepare than agarose gels. Because oxygen inhibits the polymerization process, they must be poured between glass plates (or cylinders).
Acrylamide is a potent neurotoxin and should be handled with care! Wear disposable gloves when handling solutions of acrylamide, and a mask when weighing out powder. Polyacrylamide is considered to be non-toxic, but polyacrylamide gels should also be handled with gloves due to the possible presence of free acrylamide.
Polyacrylamide gels have a rather small range of separation, but very high resolving power. In the case of DNA, polyacrylamide is used for separating fragments of less than about 500 bp. However, under appropriate conditions, fragments of DNA differing is length by a single base pair are easily resolved. In contrast to agarose, polyacrylamide gels are used extensively for separating and characterizing mixtures of proteins.
Cross section diagram of gel apparatus design:
A. Tube
B. Vertical Slab
C. Horizontal submarine
D. Horizontal thin layer with buffer pads
Principle of Separation
• According to charge
• According to size
(1) According to charge
• When charged molecules are placed in an electric field, they migrate toward either the positive (anode) or negative (cathode) pole according to their charge.
Instruments and Reagents
• Buffer
• Power supply
• Supporting media
• Detection and Quantification
I. Buffer
• Function of buffer
• 1. carries the applied current
• 2. established the pH
• 3. determine the electric charge on the solute
• High ionic strength of buffer
• produce sharper band
• produce more heat
• Commonly used buffer
• Barbital buffer & Tris-EDTA for protein
• Tris-acetate-EDTA & Tris-borate-EDTA (50mmol/L; pH 7.5-7.8)
II. Power Supply
• Function: To supply electric power
• Constant voltage (increasing of heat over time
• Heat = E x I x t
• Constant current
• according to Ohm’s law E= I x R
• R decrease over time, which inturn decrease the heat effect
E= EMF in volts (V)
I = Current in amperes (A)
t = Time in second (s)
III.Support materials used in electrophoresis
• Paper
• Starch
• Agar/agarose
• Cellulose acetate
• polyacrylamide gel
Support Media (ii)
• Agarose and polyacrylamide gels are across-linked, spongelike structure
• It is important that the support media is electrically neutral. Presence of charge group may cause:
• -Migration retardation
-The flow of water toward one or the other electrode so called ‘Electroendosmosis (EEO)’, which decrease resolution of the separation
(1) Agarose
• What is agarose?
• a chain of sugar molecules
• Extracted from seaweed
(1) Agarose gels (ii)
• For the separation of (1) large protein or protein complex (2) polynucleotide 50-30,000 base-pairs
The pore size is determined by adjusting the concentration of agarose in a gel (normally in the rank of 0.4-4% OH, O, CH2OH,
(2) Polyacrylamide gels
CH2=CHCONH2 + CH2(NHCOHC=CH2)2
Acrylamide N,N,N,N-methylenebisacrylamide
Free radical catalyst
-CH2-CH-CH2-CH-CH2-CH-
-CH2-CH-CH2-CH-CH2-CH-
CO, NH, CH2
A. Agarose gel B. Polyacrylamide gel
Polymerization of acrylamide gel can be initiated either by a chemical peroxide or by photochemical method
1. The most common method, ammonium persulphate as the initiator peroxide and TEMED as the catalyst
2. For photochemical polymerization, riboflavin and long-wave UV light are initiator and TEMED as the catalyst
Types of Detectors in HPLC
Types of Detectors in HPLC
UV/Visible Detector
The 2489 UV/Visible Detector is a versatile, dual-wavelength absorbance detector for HPLC. This detector offers the high sensitivity required for routine UV-based applications to low-level impurity identification and quantitative analysis.
The detector’s extended linear range of 2.5 AU makes for easy quantitation of both major and minor components in the same run. You can also simultaneously monitor absorbance at two user-selectable wavelengths for more information per run.
Operating as a stand-alone or as an integral component of Waters HPLC Systems, the 2489 will enhance the productivity of your chromatography laboratory. It offers a wide range of flow cell options and integrated features:
• Enhanced sensitivity via patented TaperSlit™ flow cell, which channels light through the cell for better energy throughput while minimizing RI effects
• Low noise performance (<5 µAU)
• Flexible sampling rates, from 1 to 80 Hz, for normal and fast LC separations
• Independent optimization of high-speed data rates and filter time constants allows for the accurate integration of narrow, sharp peaks
• Enhanced thermal wander management reduces baseline wander due to fluctuations caused by ambient conditions
• Accessible detector components for easy operation and maintenance
Photodiode Array (PDA) Detector
The 2998 Photodiode Array (PDA) Detector offers advanced optical detection for Waters analytical HPLC, preparative HPLC, or LC/MS system solutions. Providing unprecedented trace impurity detection and quantitation, the 2998 PDA Detector’s integrated software and optics innovations deliver high chromatographic and spectral sensitivity. Enhanced software control provides flexibility for simultaneous 2D and 3D operation with either Empower™ or MassLynx™ Software.
The 2998 PDA Detector is designed for a range of laboratory applications, including trace impurity detection and quantification, compound identification and method development.
Features:
• Patented TaperSlit™ flow cell ensures high sensitivity while maintaining optimal spectral performance
• Maximum signal-to-noise performance of <10 µAU
• Simultaneous quantification of high- and low-level components within a single chromatographic separation for extended linear range
• Flexible sampling rates for normal and fast LC separations from 1 to 80 Hz
• Definitive compound identification & co-elution detection
• Superior linear range with constant optical bandpass
• Spectral exposure optimization to maximize the signal-to-noise across the entire wavelength range with a single Deuterium lamp
• Thermal wander management for maximum baseline stability
Refractive Index (RI) Detector
The 2414 Refractive Index (RI) Detector is designed for high-performance liquid chromatography (HPLC) applications. It provides sensitivity, stability, and reproducibility for the analysis of components with limited or no UV absorption. The solution-centered design combines with Alliance® or Breeze™ HPLC Systems for RI-based HPLC and GPC applications.
The 2414 RI Detector provides solutions for preparative and analytical applications including alcohol, sugar, saccharide, fatty acid, and polymer analysis.
Features:
• Low dispersion volume for high-speed GPC and narrowbore column chromatography from 0.1 to 10 mL/min
• Thermally isolated optics bench for temperature stability and performance reproducibility
• Save solvent and maintain stability with convenient recycle valve
• Diagnostics and simplified design and control options for easy maintenance and everyday use
• Circuitry and software in the detector’s thermally isolated optics bench provide a sensitive, stable signal response to changes in refractive index independent of ambient temperature changes
• Auto-zero, auto-purge, and LED-displayed diagnostic programming enable unattended operation and to simplify methods development
Electrochemical Detector
The 2465 Electrochemical Detector combines sensitivity, reliability and simplicity for HPLC electrochemical detection. Its versatile detection modes are ideally suited for analyzing a wide range of compounds.
The 2465 Electrochemical Detector’s multiple flow-cell configuration combines working and reference electrode designs to minimize cost and enhance flexibility. The flow cell accommodates several reference electrodes: ISAAC; traditional salt-bridge design, which limits air bubble formation; and Hy-Ref, which is ideal for carbohydrate analysis and is compatible with extreme mobile phases.
The detector is designed for ultra-trace analysis in standard and microbore Alliance® HPLC based, Empower™ Software controlled applications.
Features:
• Multiple detection modes, including direct current, pulsed amperometric, and scanning
• Easier maintenance with front-mounted flow cell for simple cleaning and assembly
• Stabilized temperature for better accuracy with the integral flow cell and column heater compartment
• Faraday-shielded oven with thermal operating stability and reduced noise and drift characteristics
• 21 CFR and Part 11 compliant-ready
Conductivity Detector
The 432 Conductivity Detector provides solutions for chemically-suppressed or single-column ion chromatography. Use it as a stand-alone module or configure it into an HPLC system that links to chromatography data workstations
• High sensitivity and stability due to its multi-electrode flow cell and user-programmable temperature control, which automatically adjusts the oven temperature to minimize baseline drift
• Reduced flow path volume for narrow-bore applications
• Increased operational uptime with self-diagnostics
• Easy, unattended routine analyses with autozero and autoranging capabilities
• Sophisticated circuitry efficiently isolates detection electrodes from sources of transient electronic noise for optimal sensitivity
• Autozero feature automatically sets the baseline to zero at the start of each run eliminating the need for manual adjustments
• Autorange ensures a precise conductivity base range
• Leak sensors alerts user if leak is detected in flow cell
Fluorescence Detector
The 2475 Multi-Wavelength Fluorescence Detector is a multi-channel, tunable, fluorescence detector designed for HPLC applications. The detector delivers the highest sensitivity and selectivity, whether you are monitoring low concentrations of target compounds or low-level impurities.
Use the detector as a stand-alone unit or as an integral part of a Waters chromatography system. It operates from 200 to 900 nm and uses newly designed optics, with an enhanced illumination system, for improved performance. These design features increase the optical throughput and sensitivity resulting in an overall increase in the signal-to-noise ratio.
Features:
• Innovative flow cell designed for less dispersion, less stray light, less volume, and more pathlength for maximum sensitivity
• Three-dimensional spectral scanning for faster method development and optimization, and enhanced peak identification
• Single or multi-channel operation monitors fluorescence at one or more discrete wavelength pairs
• Axially illuminated flow cell – Allows for better light absorption resulting in highest sensitivity.
• Advanced optical design - Maximizes light throughput, and reduces light scatter, allowing for better signal-to-noise performance.
• Low noise performance - Less stray light due to orthogonal optics and the use of mirrors, not lenses, minimize scatter.
• Multiple detection modes - 2D, 3D, and on-the-fly spectral scanning – can greatly reduce the time needed to develop fluorescence detection methods by quickly determining wavelength maxima.
• Integral erbium calibration reference – Ensures wavelength accuracy.
UV/Visible Detector
The 2489 UV/Visible Detector is a versatile, dual-wavelength absorbance detector for HPLC. This detector offers the high sensitivity required for routine UV-based applications to low-level impurity identification and quantitative analysis.
The detector’s extended linear range of 2.5 AU makes for easy quantitation of both major and minor components in the same run. You can also simultaneously monitor absorbance at two user-selectable wavelengths for more information per run.
Operating as a stand-alone or as an integral component of Waters HPLC Systems, the 2489 will enhance the productivity of your chromatography laboratory. It offers a wide range of flow cell options and integrated features:
• Enhanced sensitivity via patented TaperSlit™ flow cell, which channels light through the cell for better energy throughput while minimizing RI effects
• Low noise performance (<5 µAU)
• Flexible sampling rates, from 1 to 80 Hz, for normal and fast LC separations
• Independent optimization of high-speed data rates and filter time constants allows for the accurate integration of narrow, sharp peaks
• Enhanced thermal wander management reduces baseline wander due to fluctuations caused by ambient conditions
• Accessible detector components for easy operation and maintenance
Photodiode Array (PDA) Detector
The 2998 Photodiode Array (PDA) Detector offers advanced optical detection for Waters analytical HPLC, preparative HPLC, or LC/MS system solutions. Providing unprecedented trace impurity detection and quantitation, the 2998 PDA Detector’s integrated software and optics innovations deliver high chromatographic and spectral sensitivity. Enhanced software control provides flexibility for simultaneous 2D and 3D operation with either Empower™ or MassLynx™ Software.
The 2998 PDA Detector is designed for a range of laboratory applications, including trace impurity detection and quantification, compound identification and method development.
Features:
• Patented TaperSlit™ flow cell ensures high sensitivity while maintaining optimal spectral performance
• Maximum signal-to-noise performance of <10 µAU
• Simultaneous quantification of high- and low-level components within a single chromatographic separation for extended linear range
• Flexible sampling rates for normal and fast LC separations from 1 to 80 Hz
• Definitive compound identification & co-elution detection
• Superior linear range with constant optical bandpass
• Spectral exposure optimization to maximize the signal-to-noise across the entire wavelength range with a single Deuterium lamp
• Thermal wander management for maximum baseline stability
Refractive Index (RI) Detector
The 2414 Refractive Index (RI) Detector is designed for high-performance liquid chromatography (HPLC) applications. It provides sensitivity, stability, and reproducibility for the analysis of components with limited or no UV absorption. The solution-centered design combines with Alliance® or Breeze™ HPLC Systems for RI-based HPLC and GPC applications.
The 2414 RI Detector provides solutions for preparative and analytical applications including alcohol, sugar, saccharide, fatty acid, and polymer analysis.
Features:
• Low dispersion volume for high-speed GPC and narrowbore column chromatography from 0.1 to 10 mL/min
• Thermally isolated optics bench for temperature stability and performance reproducibility
• Save solvent and maintain stability with convenient recycle valve
• Diagnostics and simplified design and control options for easy maintenance and everyday use
• Circuitry and software in the detector’s thermally isolated optics bench provide a sensitive, stable signal response to changes in refractive index independent of ambient temperature changes
• Auto-zero, auto-purge, and LED-displayed diagnostic programming enable unattended operation and to simplify methods development
Electrochemical Detector
The 2465 Electrochemical Detector combines sensitivity, reliability and simplicity for HPLC electrochemical detection. Its versatile detection modes are ideally suited for analyzing a wide range of compounds.
The 2465 Electrochemical Detector’s multiple flow-cell configuration combines working and reference electrode designs to minimize cost and enhance flexibility. The flow cell accommodates several reference electrodes: ISAAC; traditional salt-bridge design, which limits air bubble formation; and Hy-Ref, which is ideal for carbohydrate analysis and is compatible with extreme mobile phases.
The detector is designed for ultra-trace analysis in standard and microbore Alliance® HPLC based, Empower™ Software controlled applications.
Features:
• Multiple detection modes, including direct current, pulsed amperometric, and scanning
• Easier maintenance with front-mounted flow cell for simple cleaning and assembly
• Stabilized temperature for better accuracy with the integral flow cell and column heater compartment
• Faraday-shielded oven with thermal operating stability and reduced noise and drift characteristics
• 21 CFR and Part 11 compliant-ready
Conductivity Detector
The 432 Conductivity Detector provides solutions for chemically-suppressed or single-column ion chromatography. Use it as a stand-alone module or configure it into an HPLC system that links to chromatography data workstations
• High sensitivity and stability due to its multi-electrode flow cell and user-programmable temperature control, which automatically adjusts the oven temperature to minimize baseline drift
• Reduced flow path volume for narrow-bore applications
• Increased operational uptime with self-diagnostics
• Easy, unattended routine analyses with autozero and autoranging capabilities
• Sophisticated circuitry efficiently isolates detection electrodes from sources of transient electronic noise for optimal sensitivity
• Autozero feature automatically sets the baseline to zero at the start of each run eliminating the need for manual adjustments
• Autorange ensures a precise conductivity base range
• Leak sensors alerts user if leak is detected in flow cell
Fluorescence Detector
The 2475 Multi-Wavelength Fluorescence Detector is a multi-channel, tunable, fluorescence detector designed for HPLC applications. The detector delivers the highest sensitivity and selectivity, whether you are monitoring low concentrations of target compounds or low-level impurities.
Use the detector as a stand-alone unit or as an integral part of a Waters chromatography system. It operates from 200 to 900 nm and uses newly designed optics, with an enhanced illumination system, for improved performance. These design features increase the optical throughput and sensitivity resulting in an overall increase in the signal-to-noise ratio.
Features:
• Innovative flow cell designed for less dispersion, less stray light, less volume, and more pathlength for maximum sensitivity
• Three-dimensional spectral scanning for faster method development and optimization, and enhanced peak identification
• Single or multi-channel operation monitors fluorescence at one or more discrete wavelength pairs
• Axially illuminated flow cell – Allows for better light absorption resulting in highest sensitivity.
• Advanced optical design - Maximizes light throughput, and reduces light scatter, allowing for better signal-to-noise performance.
• Low noise performance - Less stray light due to orthogonal optics and the use of mirrors, not lenses, minimize scatter.
• Multiple detection modes - 2D, 3D, and on-the-fly spectral scanning – can greatly reduce the time needed to develop fluorescence detection methods by quickly determining wavelength maxima.
• Integral erbium calibration reference – Ensures wavelength accuracy.
Agarose Electrophoresis
Agarose Electrophoresis
Agarose elctrophoresis is performed to visualize your PCR products. This step allows
you to determine whether your PCR was successful, whether the resulting product is the correct size, whether other products were amplified as well, and whether the
concentration of the resulting product is suitable for cycle sequencing. All gel work
should be performed in the HIGH DNA area.
Precautions
1. Always wear gloves! This is essential to limit the spread of PCR products around
the lab as well as protecting against exposure to Ethidium Bromide.
2. Treat Ethidium Bromide with respect (it is a powerful mutagen) and ensure that it
does not get spread around the lab.
3. Wash hands when finished.
Materials Equipment
1. Agarose 1. Balance
2. 0.5X TBE 2. Erlenmeyer flask
3. weighing paper 3. Microwave
4. Gel box with comb
Step 1: Mixing Gel
1. On the scale, weigh 1 gram of agarose onto a piece of weighing paper.
2. Add agarose to Erlenmeyer flask.
3. Add 100mL of 0.5X TBE to Erlenmeyer flask.
4. Swirl vigorously to thoroughly mix agarose.
Slurry will by opaque.
Step 2: Melting Gel
1. Put agarose and 0.5x TBE slurry into microwave.
2. Heat on HIGH for 30 seconds at a time.
3. After 30 seconds, remove from microwave and swirl. Becareful as the mixtue will be HOT.
4. After 1 minute of heating, repeat heating and swirling procedure every 10 seconds until mixture is clear.
5. If the mixture begins to boil, stop and IMMEDIATELY remove the agarose. Swirl until
mixture is clear.
Step 3: Pouring the Gel
1. Before pouring, gel must be cool enough for you to hold the Erlenmeyer flask in your hand. Pouring boiling hot gel will warp the gel boxes.
2. Place gel tray into casting chamber.
3. Add casting comb(s) into the appropriate slot(s).
4. Pour agarose into gel tray to about 5-7mm.
5. Let sit for at least 30 min, until gel is cool to touch and is opaque in appearance.
6. Once set, place gel and tray into gel rig, with wells on the left (cathode) side. Then, fill gel rig with 0.5X TBE sufficient to cover the entire gel.
7. Carefully remove combs by pulling them upwardsfirmly and smoothly in a continuous motion. The remaining depressions are the wells into which your samples will be loaded.
Step 4: Loading the Gel
1. Cut a piece of parafilm and place it flat on the bench top. You may have to rub or scratch it to stick it down.
2. Using a pipette, place small dots of 6X loading dye (about 1-2uL) onto parafilm, in rows of 8, 1 dot for each PCR sample that will loaded on the gel. It is not necessary to be exact, and it is not necessary to change the tip.
3. Using a pipette take 3 μl of product and pipette it onto its corresponding dye dot, then mix the sample and dye by pipetting up and down. Then, pipette up the dye/sample solution and pipette it into the proper well.
4. After loading the first sample into the well, wash out your pipette tip by pipetting up and down several times in the buffer, then blot tip dry with a Kimwipe. In this way, all of your samples may be loaded using ONE tip.
5. Once all samples have been loaded into the gel, using a fresh tip, pipette 3uL of
the DNA ladder into well number 1 of each row of wells.
6. Put gel box cover into place (this step is essential for your gel to run and to minimize the risk of electric shock). Turn on power supply. Run the gel for 75
minutes at 80 volts.
7. Check for bubbles at the cathode side to ensure that gel rig is running. Clean up, deglove, and wash hands.
Step 5: Visualizing the Gel
1. Turn off power supply. Using gloved hands, remove the cover from the gel box.
2. Remove the gel and casting tray from rig, avoiding dripping buffer all over the
benchtop.
3. Carefully slide the gel off of the casting tray and into ethidium bromide solution.
Caution: ethidium bromide is a mutagen and should be treated with respect. If you
get ethidium bromide on you gloves, immediately change your gloves, placing the
contaminated gloves into the ethidium bromide trash. Do not spread ethidium
bromide around the lab.
4. Soad the gel in the ethidium bromide solution for 10-15 minutes to stain. Using a
spatula, remove the gel from ethidium bromide and rinse for 30 seconds in a tray
0.5x buffer. The gel may be left in the 0.5X buffer for longer to “destain” the gel
and improve contrast of stain, but this step is often unnecessary.
5. Using the spatula, carefully remove the gel from the destaining buffer and place
the gel on UV light box, avoiding creating bubbles underneath the gel.
6. Put on UV protecting face shield and turn on UV light box to see stained sample
bands and DNA ladder. Ensure that all around you are protected for UV exposure.
7. If bands are strong, take a picture using the Polaroid camera (below). If bands are
weak, return gel to ethidium bromide stain bath as the gel has been insufficiently
stained.
Step 6: Taking a picture. Remember that the gel has been soaking in Ethidium
Bromide. All objects and surfaces that the gel comes into contact with will be
contaminated with Ethidium Bromide. Always wear gloves and limit the spread of
Ethidium Bromide to protect yourself and your lab mates.
1. Ensure that you are still wearing your gloves and UV face shield.
2. With the gel flat on the UV light box, position the hood of Polaroid camera over
the gel so that the hood is centered over the gel.
3. Turn on the UV light box.
4. Keeping the camera steady, pull the trigger and HOLD the trigger until there is
silence (i.e. the shutter has opened and closed. This may take 0.5-2.0 seconds
depending on the cameras exposure settings).
5. Turn off the UV light box.
6. Pull WHITE film tab on camera to remove film. Wait 1 minute
7. After one minute has passed, pull backing off of film to check whether the gel
imaged properly.
8. Once picture is developed and you have a good picture of your gel, remove your
gel and put in gel disposal box.
9. Wipe down the UV light box with Kimwipes and dispose of in gel disposal box.
Remember that all of these surfaces and wipes are contaminated with Ethidium
Bromide.
10. Remove your gloves and dispose of in the Ethidium Bromide trash.
11. Wash hands thoroughly.
12. Label your picture.
Agarose elctrophoresis is performed to visualize your PCR products. This step allows
you to determine whether your PCR was successful, whether the resulting product is the correct size, whether other products were amplified as well, and whether the
concentration of the resulting product is suitable for cycle sequencing. All gel work
should be performed in the HIGH DNA area.
Precautions
1. Always wear gloves! This is essential to limit the spread of PCR products around
the lab as well as protecting against exposure to Ethidium Bromide.
2. Treat Ethidium Bromide with respect (it is a powerful mutagen) and ensure that it
does not get spread around the lab.
3. Wash hands when finished.
Materials Equipment
1. Agarose 1. Balance
2. 0.5X TBE 2. Erlenmeyer flask
3. weighing paper 3. Microwave
4. Gel box with comb
Step 1: Mixing Gel
1. On the scale, weigh 1 gram of agarose onto a piece of weighing paper.
2. Add agarose to Erlenmeyer flask.
3. Add 100mL of 0.5X TBE to Erlenmeyer flask.
4. Swirl vigorously to thoroughly mix agarose.
Slurry will by opaque.
Step 2: Melting Gel
1. Put agarose and 0.5x TBE slurry into microwave.
2. Heat on HIGH for 30 seconds at a time.
3. After 30 seconds, remove from microwave and swirl. Becareful as the mixtue will be HOT.
4. After 1 minute of heating, repeat heating and swirling procedure every 10 seconds until mixture is clear.
5. If the mixture begins to boil, stop and IMMEDIATELY remove the agarose. Swirl until
mixture is clear.
Step 3: Pouring the Gel
1. Before pouring, gel must be cool enough for you to hold the Erlenmeyer flask in your hand. Pouring boiling hot gel will warp the gel boxes.
2. Place gel tray into casting chamber.
3. Add casting comb(s) into the appropriate slot(s).
4. Pour agarose into gel tray to about 5-7mm.
5. Let sit for at least 30 min, until gel is cool to touch and is opaque in appearance.
6. Once set, place gel and tray into gel rig, with wells on the left (cathode) side. Then, fill gel rig with 0.5X TBE sufficient to cover the entire gel.
7. Carefully remove combs by pulling them upwardsfirmly and smoothly in a continuous motion. The remaining depressions are the wells into which your samples will be loaded.
Step 4: Loading the Gel
1. Cut a piece of parafilm and place it flat on the bench top. You may have to rub or scratch it to stick it down.
2. Using a pipette, place small dots of 6X loading dye (about 1-2uL) onto parafilm, in rows of 8, 1 dot for each PCR sample that will loaded on the gel. It is not necessary to be exact, and it is not necessary to change the tip.
3. Using a pipette take 3 μl of product and pipette it onto its corresponding dye dot, then mix the sample and dye by pipetting up and down. Then, pipette up the dye/sample solution and pipette it into the proper well.
4. After loading the first sample into the well, wash out your pipette tip by pipetting up and down several times in the buffer, then blot tip dry with a Kimwipe. In this way, all of your samples may be loaded using ONE tip.
5. Once all samples have been loaded into the gel, using a fresh tip, pipette 3uL of
the DNA ladder into well number 1 of each row of wells.
6. Put gel box cover into place (this step is essential for your gel to run and to minimize the risk of electric shock). Turn on power supply. Run the gel for 75
minutes at 80 volts.
7. Check for bubbles at the cathode side to ensure that gel rig is running. Clean up, deglove, and wash hands.
Step 5: Visualizing the Gel
1. Turn off power supply. Using gloved hands, remove the cover from the gel box.
2. Remove the gel and casting tray from rig, avoiding dripping buffer all over the
benchtop.
3. Carefully slide the gel off of the casting tray and into ethidium bromide solution.
Caution: ethidium bromide is a mutagen and should be treated with respect. If you
get ethidium bromide on you gloves, immediately change your gloves, placing the
contaminated gloves into the ethidium bromide trash. Do not spread ethidium
bromide around the lab.
4. Soad the gel in the ethidium bromide solution for 10-15 minutes to stain. Using a
spatula, remove the gel from ethidium bromide and rinse for 30 seconds in a tray
0.5x buffer. The gel may be left in the 0.5X buffer for longer to “destain” the gel
and improve contrast of stain, but this step is often unnecessary.
5. Using the spatula, carefully remove the gel from the destaining buffer and place
the gel on UV light box, avoiding creating bubbles underneath the gel.
6. Put on UV protecting face shield and turn on UV light box to see stained sample
bands and DNA ladder. Ensure that all around you are protected for UV exposure.
7. If bands are strong, take a picture using the Polaroid camera (below). If bands are
weak, return gel to ethidium bromide stain bath as the gel has been insufficiently
stained.
Step 6: Taking a picture. Remember that the gel has been soaking in Ethidium
Bromide. All objects and surfaces that the gel comes into contact with will be
contaminated with Ethidium Bromide. Always wear gloves and limit the spread of
Ethidium Bromide to protect yourself and your lab mates.
1. Ensure that you are still wearing your gloves and UV face shield.
2. With the gel flat on the UV light box, position the hood of Polaroid camera over
the gel so that the hood is centered over the gel.
3. Turn on the UV light box.
4. Keeping the camera steady, pull the trigger and HOLD the trigger until there is
silence (i.e. the shutter has opened and closed. This may take 0.5-2.0 seconds
depending on the cameras exposure settings).
5. Turn off the UV light box.
6. Pull WHITE film tab on camera to remove film. Wait 1 minute
7. After one minute has passed, pull backing off of film to check whether the gel
imaged properly.
8. Once picture is developed and you have a good picture of your gel, remove your
gel and put in gel disposal box.
9. Wipe down the UV light box with Kimwipes and dispose of in gel disposal box.
Remember that all of these surfaces and wipes are contaminated with Ethidium
Bromide.
10. Remove your gloves and dispose of in the Ethidium Bromide trash.
11. Wash hands thoroughly.
12. Label your picture.
Saturday, August 9, 2008
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