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  • User Guidelines & Standard Operating Procedurefor the

    Cary 50 UV-Vis Spectrophotometer

  • Spectrophotometer Standard Operating Procedure ii

    TABLE OF CONTENTS

    DISCLAIMER .................................................................................v

    ACKNOWLEDGEMENTS .................................................................... vi

    1. INTRODUCTION........................................................................11.1 Purpose of the Standard Operating Procedure .............................11.2 Theoretical Background........................................................1

    1.2.1 UV-Vis Theory ..............................................................11.2.2 UV-Vis Application.........................................................4

    1.3 Instrumentation .................................................................51.3.1 Instrument Accessories ...................................................6

    2. POTENTIAL HAZARDS.................................................................72.1 UV Hazard........................................................................72.2 Chemical Hazards ...............................................................7

    3. PERSONAL PROTECTIVE EQUIPMENT ...............................................8

    4. SPILL AND ACCIDENT PROCEDURES ................................................84.1 Accidents.........................................................................84.2 Spills ..............................................................................8

    5. WASTE DISPOSAL PROCEDURES ................................................... 10

    6. PROTOCOL........................................................................... 116.1 Cuvette Cleaning.............................................................. 116.2 Instrument Start-Up .......................................................... 126.3 Scan ............................................................................. 13

    6.3.1 Setting up the method parameters ................................... 136.3.2 Sample Measurement ................................................... 15

    6.4 Simple Reads .................................................................. 156.4.1 Setting up the method parameters ................................... 156.4.2 Sample measurement ................................................... 16

    6.5 Advanced Reads ............................................................... 166.5.1 Setting up the method parameters ................................... 166.5.2 Sample measurement ................................................... 17

    6.6 Kinetics ......................................................................... 176.6.1 Setting up the method parameters ................................... 176.6.2 Kinetic measurement without using the RX accessory ............. 196.6.3 Setting up the RX accessory............................................ 196.6.4 Sample measurement ................................................... 206.6.5 Cleaning the RX Accessory ............................................. 21

    6.7 Concentration ................................................................. 216.7.1 Setting up the method parameters ................................... 21

  • Spectrophotometer Standard Operating Procedure iii

    6.7.2 Sample and standard measurement .................................. 236.8 RNA/DNA ....................................................................... 23

    6.8.1 Setting up the method parameters ................................... 236.8.2 Sample Measurement ................................................... 24

    6.9 Thermal Not currently available on the Cary 50 ....................... 256.10 Enzyme Kinetics ............................................................... 25

    6.10.1 Setting up the method parameters ................................... 256.10.2 Sample measurement ................................................... 26

    6.11 Scanning Kinetics ............................................................. 276.11.1 Setting up the method parameters ................................... 276.11.2 Sample measurement ................................................... 28

    6.12 General Analysis............................................................... 296.13 Instrument Shut-down........................................................ 29

    7. QUALITY CONTROL ................................................................. 307.1 Instrument Validation ........................................................ 307.2 Lamp Alignment and Calibration ........................................... 30

    8. TROUBLESHOOTING ................................................................ 318.1 General ......................................................................... 318.2 Starting a run .................................................................. 328.3 Data collection ................................................................ 338.4 Graphics ........................................................................ 33

    8.4.1 General.................................................................... 338.4.2 Graph Labeling........................................................... 34

    8.5 File Handling................................................................... 358.6 Exporting Data................................................................. 368.7 Maths Calculator .............................................................. 378.8 User Data Form................................................................ 388.9 Recalculation and Reports................................................... 38

    9. PREVENTATIVE MAINTENANCE .................................................... 399.1 Three Months .................................................................. 399.2 As Required .................................................................... 39

    10. QUICK REFERENCE GUIDE ....................................................... 4010.1 Scan ............................................................................. 4010.2 Sign the logbook............................................................... 4010.3 Simple Reads .................................................................. 4110.4 Advanced Reads ............................................................... 4210.5 Kinetics ......................................................................... 43

    10.5.1 Kinetic measurement without using the RX accessory ............. 4410.5.2 Kinetic measurement using the RX accessory ....................... 44

    10.6 Concentration ................................................................. 4510.7 RNA/DNA ....................................................................... 4610.8 Enzyme Kinetics ............................................................... 47

  • Spectrophotometer Standard Operating Procedure iv

    10.9 Scanning Kinetics ............................................................. 48

    11. REFERENCES ...................................................................... 49

    APPENDIX 1: UV-VIS SPECTROPHOTOMETER LOG.................................... 50APPENDIX 2: MAINTENANCE LOG....................................................... 52

  • Spectrophotometer Standard Operating Procedure v

    DISCLAIMERThe materials contained in this document have been compiled from sources believed to be reliable and to represent the best opinions on the subject. This document is intended to serve only as a starting point for good practices and does not purport to specify minimal legal standards. No warranty, guarantee, or representation is made by Laurier as to the accuracy or sufficiency of information contained herein, and Laurier assumes no responsibility in connection therewith.

  • Spectrophotometer Standard Operating Procedure vi

    Issued: July 2007 Revision: 0

    ACKNOWLEDGEMENTSThe following individuals of Laurier contributed to the writing, editing, and production of this manual: Gena Braun (Instrumentation Technician); Stephanie Kibbee (Environmental/Occupational Health and Safety Office).

    This manual was prepared for Laurier. Any corrections, additions or comments should be brought to the attention of the Instrumentation Technician at 519-884-0710 ext. 2361.

  • Spectrophotometer Standard Operating Procedure 1

    1. INTRODUCTION

    1.1 Purpose of the Standard Operating Procedure

    This standard operating procedure (SOP) is NOT a substitute for training and/or reading the appropriate manuals before use. All principle investigators and supervisors must document that training has been received by students and staff who will be using the Cary 50 UV-Vis spectrophotometer.

    A list of authorized users will be kept by the Instrumentation Technician in SR314A.

    This SOP is intended to promote consistent and safe use of Cary 50 Uv-Vis spectrophotometer within the Faculty of Science. This SOP covers the potential hazards, personal protection requirements, spill and accident procedures, waste disposal considerations, and instrument operation for the Cary 50 Uv-Vis spectrophotometer (henceforth referred to as the spectrophotometer).

    1.2 Theoretical Background

    1.2.1 UV-Vis TheoryUltraviolet and visible (UV-Vis) absorption spectroscopy is the measurement of light absorption by a sample. This absorption or attenuation can occur when light passes through a translucent liquid sample, or when light is reflected from a sample surface. The difference in the incident light and the transmitted light is used to determine the actual absorbance.

    When an atom or molecule absorbs energy, electrons are promoted from their ground state to an excited state. Molecules can only absorb radiant energy in definite units, or quanta, which correspond to the energy difference between the ground and excited states. The energy, E, carried by any one quantum is proportional to its frequency of oscillation, that is:

    hc

    hE where is the frequency, the related wavelength and h = Planck's constant (6.624 x 10-27 ergs/seconds).

    In addition to electronic excitation, the atoms within a molecule can rotate and vibrate with respect to each other. These vibrations and rotations also have discrete energy levels, which can be considered as being packed on top of each electronic level. Absorption of ultraviolet and visible radiation in organic molecules is restricted to certain functional groups (chromophores) that

  • Spectrophotometer Standard Operating Procedure 2

    contain valence electrons of low excitation energy. The spectrum of a molecule containing these chromophores is complex as the superposition of atomic rotational and vibrational transitions on the electronic transitions gives a combination of overlapping lines. This appears as a continuous absorption band.

    The visible region of the spectrum comprises photon energies of 36 to 72 kcal/mol, and the near ultraviolet region, out to 200 nm, extends this energy range to 143 kcal/mol. Ultraviolet radiation having wavelengths less than 200 nm is difficult to handle, and is seldom used as a routine tool for structural analysis. UV-Vis light causes primarily electronic excitation by promoting the outer electrons of lower orbitals to higher energy levels, and is sometimes called electronic spectroscopy. The easily accessible part of this region (wavelengths of 200 to 800 nm) shows absorption only if conjugated pi-electron systems are present. There are a number of possible electronic transitions, as shown in Figure 1-1 (summarized from Sheffield Hallam University):

    to * TransitionsAn electron in a bonding orbital is excited to the corresponding antibonding orbital. The energy required is large. Absorption maxima due to to * transitions are not seen in typical UV-Vis. spectra (200 - 700 nm).

    n to * TransitionsSaturated compounds containing atoms with lone pairs (non-bonding electrons) are capable of n to * transitions. These transitions usually need less energy than to * transitions. They can be initiated by light whose wavelength is in the range 150 -250 nm. The number of organic functional groups with n to * peaks in the UV region is small.

    n to * and to * TransitionsMost absorption spectroscopy of organic compounds is based on transitions of n or electrons to the * excited state. This is because the absorption peaks for these transitions fall in an experimentally convenient region of the spectrum (200 - 700 nm). These transitions need an unsaturated group in the molecule to provide the electrons.

  • Spectrophotometer Standard Operating Procedure 3

    n**

    n**

    *

    *

    * (anti-bonding)

    * (anti-bonding)

    n (non-bonding)

    (bonding)

    (bonding)

    EN

    ER

    GY n*

    *

    n**

    *

    *

    * (anti-bonding)

    * (anti-bonding)

    n (non-bonding)

    (bonding)

    (bonding)

    EN

    ER

    GY

    Figure 1-1: Possible electronic transitions (adapted from Reusch, 1999).

    Of the six transitions illustrated, only the two of lowest energy (left-most, colored blue) are achieved by the energies available in the 200 to 800 nm spectrum. As a rule, energetically favored electron promotion will be from the highest occupied molecular orbital to the lowest unoccupied molecular orbital, and the resulting species is called an excited state. The factors that influence transition probabilities are complex, and are treated by "Selection Rules". One factor that influences these Selection Rules is the overlap of the orbitals involved in the electronic excitation. This is illustrated in Figure 1-2 by the two common transitions of an isolated carbonyl group. The n to * transition is lower in energy ( max=290 nm) than the to * transition ( max=180 nm), but the molar absoptivity (, discussed in greater detail in section 1.2.2) of the former is a thousand times smaller than the latter. The spatial distribution of these orbitals suggests why this is so. As illustrated in the diagram, the n-orbitals do not overlap at all well with the * orbital, so the probability of this excitation is small. The to * transition, on the other hand, involves orbitals that have significant overlap, and the probability is near 1.0.

    C O OC

    electron

    orbital

    n orbital

    * orbital

    C O OC

    electron

    orbital

    n orbital

    * orbital

    Figure 1-2: and n orbitals of a carbonyl group (adapted from Adapted from Reusch, 1999).

  • Spectrophotometer Standard Operating Procedure 4

    The probability of transitions is also strongly influenced by bond conjugation. Increased conjugation brings the highest occupied and lowest unoccupied molecular orbitals closer together. The energy (E) required to effect the electron promotion is therefore less, and the wavelength that provides this energy is increased correspondingly. Conjugation of double and triple bonds shifts the absorption maximum to longer wavelengths, and extending conjugation generally results in increased bathochromic (longer wavelength) and hyperchromic (greater absorbance) shifts in absorption.

    The solvent in which the absorbing species is dissolved also has an effect on the spectrum of the species. Peaks resulting from n to * transitions are shifted to shorter wavelengths (blue shift) with increasing solvent polarity. This arises from increased solvation of the lone pair, which lowers the energy of the n orbital. Often, the reverse (i.e. red shift) is seen for to * transitions. This is caused by attractive polarization forces between the solvent and the absorber, which lower the energy levels of both the excited and unexcited states. This effect is greater for the excited state, and so the energy difference between the excited and unexcited states is slightly reduced, resulting in a small red shift. This effect also influences n to * transitions but is overshadowed by the blue shift resulting from solvation of lone pairs.

    1.2.2 UV-Vis ApplicationWhen sample molecules are exposed to light having an energy that matches a possible electronic transition within the molecule, some of the light energy will be absorbed as the electron is promoted to a higher energy orbital. An optical spectrometer records the wavelengths at which absorption occurs, together with the degree of absorption at each wavelength. The resulting spectrum is presented as a graph of absorbance (A) versus wavelength.

    The UV-Vis spectra have broad features that are of limited use for sample identification but are very useful for quantitative measurements, such as concentration determination, RNA/DNA analysis, or the assessment of consumer products. Different molecules absorb radiation of different wavelengths. An absorption spectrum will show a number of absorption bands corresponding to structural groups within the molecule. UV-Vis spectroscopy is usually applied to molecules or inorganic complexes in solution and absorption measurements can be at a single wavelength or over an extended spectral range.

    The concentration of an analyte in solution can be determined by measuring the absorbance at a given wavelength and applying the Beer-Lambert Law, as follows:

    bcA

    where is a constant of proportionality, called the molar absorbtivity. Absorbance is therefore directly proportional to the path length, b (cm), and

  • Spectrophotometer Standard Operating Procedure 5

    the concentration, c (mol/L), of the absorbing species. Because the absorbance of a sample will be proportional to the number of absorbing molecules in the spectrometer light beam (e.g. their molar concentration in the sample tube), it is necessary to correct the absorbance value for this and other operational factors if the spectra of different compounds are to be compared in a meaningful way. The molar absorptivity is particularly useful when comparing the spectra of different compounds and determining the relative strength of light absorbing functions (chromophores).

    UV-Vis spectroscopy is also useful to characterize the absorption, transmission, and reflectivity of a variety of technologically important materials, such as pigments, coatings, windows, and filters. This more qualitative application usually requires recording at least a portion of the UV-Vis spectrum for characterization of the optical or electronic properties of materials.

    1.3 InstrumentationThe Cary 50 spectrophotometer features a unique design that uses a Xenon flash lamp as the source of UV-Vis radiation. This offers many advantages over traditional and diode array UV-Vis spectrophotometers. The Cary 50 is as fast as a diode array; however unlike a diode array, the Xenon lamp only flashes when it is acquiring a data point. This lessens the exposure of the sample to the entire wavelength range and so reduces the degradation of photosensitive samples. The Xenon lamp is unaffected by room light. This allows you to measure large samples with the lid off. If you choose to run an analysis with the lid open, you MUST WEAR appropriate safety glasses.

    The light flashes from the Xenon lamp are very intense, so they can be passed through a beam splitter to give simultaneous reference beam correction without causing excess photometric noise. This means that the wavelength shift errors associated with traditional scanning methods are eliminated.

    The Schwartzchild collector mirrors collect the light energy from the Xenon Flash Lamp module. This light is then focused through a lens onto the excitation entry slit. The light passes though an excitation monochrometer and into the sample where some of the light is absorbed. The remaining like is collected by a photomultiplier. The difference between the incident light and the transmitted light represents the UV absorbance of the sample (Figure 1-3).

  • Spectrophotometer Standard Operating Procedure 6

    Figure 1-3: The Cary 50 optics.

    1.3.1 Instrument Accessories

    1.3.1.1 Rapid Mix AccessoryThe RX 2000 Rapid Mix accessory from Applied Photophysics is a stop flow accessory which allows the user to start measuring reagents as soon as they have been mixed. This accessory enables the user to monitor the course of a reaction by measuring the light absorption of the initial reagents, intermediates, or end products. This technique is useful for monitoring oxidation and reduction reactions, metal-ligand complexes, salvation, dye binding, enzyme kinetics, ion interaction, enzyme conformation and denatuartion, and cell membrane studies. The RX 2000 is especially advantageous for reactions which are short-lived.

    The syringes are mounted on a rigid drive platform ensuring that the flow is stopped precisely and instantaneously. This platform and the syringe mounting blocks are extremely resistant to chemical attack. Reagents travel in the inert sample circuit through an umbilical cord to the flow cell where they are mixed by a high efficiency T-format mixer. The reagent handling system is connected to a thermostated bath and includes a window which allows the user to check for air bubbles in the drive syringes, and provides assistance when making a syringe change.

    The Rapid Mix accessory can be operated manually or using a pneumatic drive. Contact the Instrumentation Technician to use the Rapid Mix Accessory.

  • Spectrophotometer Standard Operating Procedure 7

    CAUTION!ULTRAVIOLET

    LIGHT HAZARD

    Do not open the cover when the lamp is turned on.

    Wear appropriate eye protection.

    1.3.1.2 Peltier Water Bath AccessoryThe Cary Temperature Probe accessory accurately measures temperature, in degrees Celsius, anywhere in the sample compartment. It may also be fitted to the front of the Eclipse instrument for remote temperature monitoring.

    The accessory consists of a probe box which is plugged into the accessory controller board. The probe box is fitted with two probes, consisting of platinum resistors, fitted with ceramic sleeves. Two types of probe holders are also supplied: one designed for use with rectangular cells, and one designed for use with microcells. Note: Temperature probe holders for square top and stoppered semi-microcells are supplied with the Temperature Probe accessory.

    The accessory would generally be used in conjunction with a thermostatted cell holder, to monitor the temperature inside the cells. However, the accessory may be used anytime accurate temperature readings are required. Contact the Instrumentation Technician to use the Peltier Accessory.

    2. POTENTIAL HAZARDS

    2.1 UV Hazard

    Hazardous UV radiation is emitted by the xenon flashlamp in the instrument. This radiation can cause seriousdamage to your eyes. NEVER look directly at the UVsource lamp; the lid should always be shut during dataacquisition. Wear safety glasses manufacturedto an approved standard and which are certified orotherwise warranted to protect your eyes from UVradiation if the lamp is operating and the samplecompartment must be open.

    2.2 Chemical HazardsUse of the Cary 50 system and accessories may involve materials, solvents and solutions, which are flammable, corrosive, toxic or otherwise hazardous. Careless, improper, or unskilled use of such materials can create explosion hazards, fire hazards, toxicity and other hazards which can result in death, serious personal injury, and damage to equipment and property.

    ALWAYS ensure that laboratory safety practices governing the use, handling and disposal of such materials are strictly observed. These safety practices should include the wearing of appropriate safety clothing and safety glasses.

    The Cary 50 can be connected to a RX Rapid Mixing Accessory. The RX Rapid Mixing Accessory is in turn connected to the Neslab Circulating Water Bath. This water bath contains a small amount of Chloramine-T, which is hazardous. Contact the Instrumentation Technician if the solution in the water bath is low.

  • Spectrophotometer Standard Operating Procedure 8

    3. PERSONAL PROTECTIVE EQUIPMENT

    Use of the Cary 50 requires safety glasses manufactured to an approved standard and which are certified or otherwise warranted to protect the eyes from UV radiation. A lab coat, appropriate gloves, and shoes with a closed toe and heel should also be used to provide protection from the substance to be analyzed.

    See the WLU Laboratory Health and Safety Manual for additional information on personal protective equipment: http://www.wlu.ca/documents/23120/Laboratory_Health_%26_Safety_Manual__Feb_2007_Final.pdf.

    4. SPILL AND ACCIDENT PROCEDURES

    4.1 AccidentsAll incidents are to be reported to the Instrumentation Technician and if applicable, a students supervisor.

    All accidents, incidents and near misses must also be reported to the Environmental / Occupational Health and Safety (EOHS) Office via the WLU Employee Accident / Incident / Occupational Disease Report form (www.wlu.ca/eohs/forms). To meet regulatory requirements, these forms must be submitted to EOHS within 24 hours of occurrence, with the exception of critical injuries, which must be reported immediately to the EOHS Office by telephone. Critical injuries include any of the following; place life in jeopardy, produce unconsciousness, result in substantial loss of blood, involve fracture of a leg or arm but not a finger or toe, involve amputation of a leg, arm, hand or foot, but not a finger or toe, consist of burns to a major portion of the body, or cause the loss of sight in an eye.

    Additional details regarding incident reporting can be found in the WLU Accident Incident Procedure (www.wlu.ca/eohs).

    4.2 SpillsAny spills in the sample compartment should be wiped up immediately as well as any deposits on the sample compartment windows. The exterior surfaces of the Cary 50 spectrophotometer should be kept clean.

    All cleaning should be done with a soft cloth. If necessary, this cloth can be dampened with water or a mild detergent. Do not use organic solvents or abrasive cleaning agents.

  • Spectrophotometer Standard Operating Procedure 9

    The WLU Laboratory Health and Safety Manual provides detailed instructions for dealing with major and minor spills. Do not attempt to clean up a spill if you have not been properly trained, or if you are unsure of the proper procedures. Before using ANY hazardous materials, make sure you understand the proper clean-up procedure. The Environmental/Occupational Health and Safety Office is also available to provide guidance at ext. 2874. The guidelines below are summarized from the WLU Laboratory Health and Safety Manual.

    Determine if the spill is a major or minor spill (see Table 4-1). 1. For major spills:

    a. Evacuate the lab, close the doors, restrict the area, and notify others in the area of spill, including your supervisor and the Instrumentation Technician if possible.

    b. Call ext 3333 (Community Safety and Security).c. Activate the fire alarm if there is risk to the safety of other people in the

    building.d. Be available to provide technical information to emergency responders.

    2. For minor spills:a. Attend to injured or contaminated personnel.b. Restrict the area and notify others in the lab of the spill, including your

    supervisor and the Instrumentation Technician if possible.c. Take action to minimize the extent of the spill.d. If flammable material is involved, turn of ignition sources (power, Bunsen

    burners).e. Select and wear all appropriate personal protective equipment.f. It is the responsibility of the user of the hazardous material to clean up

    the spill if he/she feels it is safe to do so.g. All personal protective equipment must be disposed of correctly, and

    must not be worn outside the laboratory.h. Apply spill pillow/pads or other absorbent material, first around the

    outside of the spill, encircling the material, then absorb to the center of the spill.

    i. Dispose of all materials used to clean up the spill in a sealed container.j. Label and dispose of all bags or containers as hazardous waste.

    3. For chemical spills on the body:a. Remove all contaminated clothing.b. Flood exposed area with running water form a safety shower for at least

    15 minutes.c. Have another individual contact 9-911 and ext 3333 to obtain medical

    attention.d. Report the incident to your supervisor and the Instrumentation

    Technician.4. For chemicals splashed in the eye(s):

    a. Immediately rinse eyeball and inner surface of eyelid with water continuously for 15 minutes. Forcibly hold eye lid(s) open to ensure effective wash behind eyelids.

  • Spectrophotometer Standard Operating Procedure 10

    b. Have another individual contact 9-911 and ext 3333 to obtain medical attention.

    5. Report the incident to your supervisor and the Instrumentation Technician.

    Table 4-1: Guidelines for classification of a major spill

    Material Quantity

    Air and water reactive materials All quantitiesFlammable liquids Greater than 4LCombustible liquids Greater than 4LNon-flammable organic liquids Greater than 4L

    Concentrated acids Liquids greater than 1LSolids greater than 1kg

    Concentrated bases and alkalis Liquids greater than 1LSolids greater than 1kg

    Mercury Greater than 30 ml

    Oxidizers Liquids greater than 1LSolids greater than 500g

    Highly toxic, highly malodorous material

    Liquids greater than 100 mlSolids greater than 50g

    Low hazard material At the discretion of laboratory personnel

    Compressed gas leaks If the leak cannot be stopped by closing the valve on the gas cylinder

    5. WASTE DISPOSAL PROCEDURESIf any hazardous chemicals are used for sample analysis or preparation, they must be disposed of properly, as outlined in the WLU Laboratory Health and Safety Manual.

  • Spectrophotometer Standard Operating Procedure 11

    6. PROTOCOL

    6.1 Cuvette Cleaning1. Select the correct type of cuvette for your analysis:

    a. Quartz cuvettes should be used for readings in the UV range (200-350 nm).

    b. Disposable (plastic) cuvettes can be used for readings in the visible range (350-750 nm).

    c. Cuvettes are supplied by each individual/lab using the spectrophotometer, and users are responsible for ensuring that cuvettes are properly cleaned and stored.

    2. Dirty cells are the greatest single source of error in spectrophotometry. Avoid handling the cells by the polished surfaces.

    3. Cuvettes need to be cleaned properly both before and after use. The cleaning procedure for glass or quartz cuvettes is dependent on the sample that was measured, so cleaning is sample specific. The smaller and more dilute the sample being measured, the more important the cleanliness of the cuvette. Regardless, the following advice should be followed: a. Rinse the cuvette with the same dispersant that was used for the

    measurement, i.e. if the sample was dispersed in water use clean water to rinse it.

    b. Clean the cuvette in an ultrasonic bath of clean solvent. Rinse with 5-10 volumes of 18.2M ohm Milli-Q water. Do not use acetone. Do not clean with hydrofluoric acid which will attack quartz. Do not clean with strongly alkali solutions.

    c. Solution spillage should never be allowed to dry on the cells and should be wiped off with a kimwipe or lint free cloth.

    d. Once clean, dry the cuvette with nitrogen. 4. Common contaminants on cuvette surfaces include:

    a. Synthetic detergent solutions; Most commercial preparations are strongly fluorescent. Before use, the fluorescence characteristics of a dilute solution of the detergent should be measured to make quite sure that the fluorescence is minimal at the chosen analytical wavelengths.

    b. The growth of micro-organisms in buffer or reagent solutions will affect blank values by both their absorbance and light scattering properties.

    c. Filter paper can be another source of contamination due to residues of phenols present from the original wood.

  • Spectrophotometer Standard Operating Procedure 12

    6.2 Instrument Start-Up1. Sign the logbook.2. Turn on the computer (the Cary 50 will start automatically, and run start-up

    diagnostics).3. Cancel the Novell Login (twice).4. Let the instrument warm up for approx 15 minutes before starting

    measurements.5. Double click on the Cary icon on the desktop.6. See Table 6-1 for a description of the applications available on the

    spectrophotometer.

    Table 6-1: Applications available on the spectrophotometer

    Application Description

    ScanThis application enables you to scan samples across a wavelength or wavenumber range and manipulate the collected data.

    Simple ReadsThe Simple Reads application is used to perform simple absorbance readings of samples.

    Kinetics The Kinetics application is used to calculate reaction rates from absorbance versus time data.

    ConcentrationThe Concentration application is used to determine the concentration of an absorbing sample, using up to a 30-point calibration for quantitative analysis.

    Advanced Reads

    Advanced Reads: The Advanced Reads application allows you set up methods to read multiple samples in a single run. The features of this application include: finding the mean of multiple readings of the sample solution; four different ordinate modes (absorbance, percent transmittance, Abs*F and percent reflectance); and, the ability to use various accessories.

    Thermal

    The Thermal application allows you to perform thermal analyses on DNA using one of the thermostattable Cary accessories. This application is NOT CURRENTLY AVAILABLE ON THE CARY 50 in SR416.

    RNA/DNARNA/DNA: The RNA-DNA application can be used to collect data and calculate the absorbance and absorbance ratios used in determining the amount, type and purity of

  • Spectrophotometer Standard Operating Procedure 13

    nucleic acid samples

    Table 6-1 continued: Applications available on the spectrophotometer

    Application Description

    Enzyme KineticsThe Enzyme Kinetics application allows you to perform an Enzyme Kinetics run and perform calculations on your results using the Michaelis-Menten equation.

    Scanning Kinetics

    The Scanning Kinetics application allows you to scan samples across a wavelength or wavenumber range. From the resultant absorbance versus wavelength data, an absorbance versus time (kinetics) curve can be obtained for any wavelength in the range.

    Multi

    Varian's Multicomponent application is a powerful multivariate analysis tool that allows users to develop strong and reliable calibration models and use them to accurately predict the concentration of unknown samples. You can collect new scans or use existing scans to make your models.

    ColorThe Cary Color Calculations application is a module which allows you to perform calculations on data collected by Cary spectrophotometers

    Maths The Maths window enables you to perform mathematical manipulations on collected traces.

    6.3 Scan

    6.3.1 Setting up the method parameters1. NOTE: A detailed description of all of the set-up parameters is listed on the

    Cary 50 Help and Videos CD.2. Select Scan in the Cary WinUV menu. 3. Set the method for the analysis: Select File/Open Method from the menu to

    load a previously defined method and skip to Section 6.3.2. If you dont have a method, select the Setup button to display the Setup dialog and specify the method parameters for a new method by following steps 4 through 9.

    4. Under the Cary tab set the instrument parameters as follows:a. Set the wavelength range for the scan by entering the values you require

    in the Start/Stop fields, for example 500/400. b. In the Y Mode group select the ordinate Mode in which you want the

    collect data to be displayed, for example Abs. Enter an upper range and

  • Spectrophotometer Standard Operating Procedure 14

    lower range value in the Y min and Y max entry fields to specify the displayed ordinate range.

    c. Make sure that Cycle Mode is not selected. d. Set the Beam Mode for the run. This is should be set to 'Dual Beam'. e. In the Scan Controls group, select Simple and click a scan speed button.

    Alternatively, you can select Advanced and enter an Ave Time and Data Interval. (The Cary will then select the Scan Rate).

    f. In the Display Options group, select the way in which you want the data displayed as it is collected. Choose Individual Data to display the collected data of each sample in individual graph boxes. Choose Overlay Data to superimpose the collected data of each sample in the Scan run in one graph box.

    g. Select the Status Display check box so that you can view various instrument parameters during the scan to setup visual system monitoring.

    5. Set up baseline correction under the Baseline tab:a. Select Baseline Correction. This will force the Cary to perform a baseline

    correction on the sample data. The correction will be performed on each point before it is displayed.

    - Note: You can use a stored baseline at this stage. To do so, press the Retrieve Baseline button and open the saved CSW baseline file. However, it is recommended that a new baseline be generated for each analysis.

    6. Select the Accessories 1 and Accessories 2 tabs and make sure that no options are selected and no accessories are installed.

    7. There are no selectable options on the Samplers tab.8. Set up reporting and printing requirements under the Reports tab:

    a. Enter your name in the Name entry field. b. Enter a comment relating to your experiment in the Comment entry field. c. Set up your report style by selecting the appropriate check boxes in the

    Options group, e.g.:- Select the AutoPrint check box to obtain a printout of your report

    automatically.- Select the Parameters check box to include your method parameters

    in the report. - Select the Graph check box to include a graph in the generated

    report. d. Set up the Peak Table reporting.

    - Press the Peak Information button and choose the type of Peak Labels, the Peak Style and set the Peak Threshold. Press OK.

    - Select Maximum Peak to report the peak with the largest peak threshold that exceeds the Peak Threshold value.

    - Select All Peaks to report all peaks meeting the Peak Style criterion and exceeding the Threshold value.

    e. Set up X-Y pairs reporting if desired. You can use the actual Data Interval by which the data was collected or you can make the Cary interpolate the points to a new Interval.

    f. Select the Autoconvert option (if desired)

  • Spectrophotometer Standard Operating Procedure 15

    - If you select for ASCII (csv) or Select for ASCII (csv) with Log, then at the end of the data collection the system will automatically generate a report and store the data both in the Cary format as well as ASCII XY pairs format in the current folder.

    9. Set up storage of collected data before run under the Auto Store tab:a. Select Storage On (Prompt at Start).

    10.Finish Setup -- Once you are satisfied with your method setup select OK to confirm any changes you have made and close the Setup dialog. Save the method if you plan to use it regularly.

    6.3.2 Sample Measurement1. Zero the instrument by clicking on the zero button. Load the blank, and

    press OK.2. Click the Baseline button to set up to baseline collection system.

    a. When prompted, place the blank solution or empty cuvette in the sample compartment. Make sure not to touch the side of the cuvette while doing so. Press OK.

    b. After the collection, the word 'baseline' will appear in red in the ordinate status box, indicating that you are in baseline correction mode and you have a valid baseline file for the correction.

    3. Click the Start button to start a data collection.4. Once you press Start, the Windows Save As dialog will appear. Enter the

    appropriate name for your Scan run and press Save. 5. The Sample Name dialog will now appear. Enter the appropriate name for

    you sample and press OK. The Scan run will commence and the corrected trace will appear in the Graphics area.

    6. Save the collected data -- Once the run is finished, select the Save As command from the File menu. In the File name field, enter a file name for this scan run.

    6.4 Simple Reads

    6.4.1 Setting up the method parameters1. NOTE: A detailed description of all of the set-up parameters is listed on the

    Cary 50 Help and Videos CD.2. Select Simple Reads. 3. Set the method for the analysis: Select File/Open Method from the menu to

    load a previously defined method and skip to Section 6.4.2. If you dont have a method, select the Setup button to display the Setup dialog and specify the method parameters for a new method by following steps 4 and 5.

    4. Under the Cary tab set the instrument parameters as follows:a. Select 'Read at Wavelength' and enter the wavelength at which you want

    to perform the read.

  • Spectrophotometer Standard Operating Procedure 16

    b. In the Y Mode group select the button corresponding to the ordinate mode you require. The ordinate mode determines the way in which the photometric value is measured and displayed in your report.

    c. Click the OK button. The instrument will change to the new wavelength.5. Finish Setup -- Once you are satisfied with your method setup select OK to

    confirm any changes you have made and close the Setup dialog. Save the method if you plan to use it regularly.

    6.4.2 Sample measurement1. Zero the instrument.

    a. Place the blank solution or empty cuvette in the sample compartment of the Cary Eclipse instrument. Make sure not to touch the side of the cuvette while doing so.

    b. Click the Zero button to zero the system. Alternatively, select Zero fromthe Commands menu to perform a zero.

    2. Put your sample into the cell holder and press the Read button. The instrument will take measurements and the results will appear in the Report area (bottom box).

    3. Save the collected data -- Once the run is finished, select the Save As command from the File menu. In the File name field, enter a file name for this scan run.

    6.5 Advanced Reads

    6.5.1 Setting up the method parameters1. NOTE: A detailed description of all of the set-up parameters is listed on the

    Cary 50 Help and Videos CD.2. Select Advanced Reads. 3. Set the method for the analysis: Select File/Open Method from the menu to

    load a previously defined method and skip to section 6.5.2. If you dont have a method, select the Setup button to display the Setup dialog and specify the method parameters for a new method by following steps 4 through 8.

    4. Under the Cary tab set the instrument parameters as follows:a. In the Wavelength field, enter the wavelength that you want to monitor.b. In the Ave. time field, set the amount of time, in seconds, for which data

    is averaged. Average time is automatically set for 0.1000 seconds but can be adjusted if needed.

    c. Select Replicates or Sample Averaging. For Replicates, enter the number of replicates of each sample that you would like read. For Sample Averaging, enter '2' for duplicate aliquots of the same solution or '3' for triplicate aliquots.

    d. Select the ordinate mode you require from the drop down list in the Y Mode field. Enter a Factor value if you have selected Abs*F.

    e. Select the Show Status Display check box on any of the tabs to display various information fields on your current reaction.

  • Spectrophotometer Standard Operating Procedure 17

    5. Select the Samples tab. This dialog box allows you to enter a list of sample names that will be used during your analysis.

    a. Enter the number of samples that you are going to use in the Number of Samples field. The table below this field will expand or contract to match your choice.

    b. In the Samples table (in the Sample Names group), enter the name of each sample. You can enter up to 20 characters for each name.

    c. If the samples have the same name with a different numeric extension, enter the name in the first sample position and then press the Increment button.

    6. Under the Accessories tab, select the accessories to use in the analysis:a. Select the Accessories 1 and Accessories 2 tabs and make sure that no

    accessories are selected.7. Set up reporting and printing requirements as desired (see section 6.3.1

    steps 8 and 9 for details).8. Finish Setup -- Once you are satisfied with your method setup select OK to

    confirm any changes you have made and close the Setup dialog. Save the method if you plan to use it regularly.

    6.5.2 Sample measurement1. Place a blank in the sample compartment and select the Zero button from

    the application window. Load the blank and press OK. 2. Click the Start button. The Sample Selection dialog box will appear. 3. Select the samples you would like to read then click OK. 4. Enter a file name and click OK to save the data.5. The Present Sample dialog box will prompt you to place the appropriate

    sample in the sample compartment. Load the sample and press OK to read. (If replicates have been chosen, then the result is reported after the final sample replicate is read.)

    6. Repeat for the remaining samples. 7. Save your results.

    6.6 Kinetics

    6.6.1 Setting up the method parameters1. NOTE: A detailed description of all of the set-up parameters is listed on the

    Cary 50 Help and Videos CD.2. Select the Cary Icon from the Desktop and choose Kinetics3. Set the method for the analysis: Select File/Open Method from the menu to

    load a previously defined method and skip to Section 6.6.2. If you dont have a method, select the Setup button to display the Setup dialog and specify the method parameters for a new method by following steps 4 though 10.

    4. Under the Cary tab set the instrument parameters as follows:a. In the Wavelength field, enter the wavelength that you want to monitor.

  • Spectrophotometer Standard Operating Procedure 18

    b. In the Ave. time field, set the amount of time, in seconds, for which data is averaged. Average time is automatically set for 0.1000 seconds but can be adjusted if needed.

    c. Enter an upper range and lower range value in the Y min and Y max entry fields to specify the displayed ordinate range.

    d. Select the abscissa (X mode) you require. Click Min to time in minutes or Sec to time in seconds.

    e. Select the Advanced Collect radio button in the Collect Timing group. This enables you to set up different data collection procedures for the multiple rates in your reaction.

    - Enter the number of different reaction rates that you require in the Number of Stages entry field. The number you set here will be reflected in the table below.

    - Specify how long the Cary will wait after reading each cell before it starts another reading cycle by setting the Cycle time for each rate Stage.

    - Specify the duration of the measurement run by setting the Stop time for each rate Stage.

    f. Select the Show Status Display check box on any of the tabs to display various information fields on your current reaction.

    5. Under the Options tab, set up the Kinetics options:a. In the Display Options group, select the way in which you want the data

    displayed as it is collected. Choose Individual Data to display the collected data of each sample in individual graph boxes. Choose Overlay Data to superimpose the collected data of each sample in the Scan run in one graph box.

    6. Under the Accessories tab, select the accessories to use in the analysis:a. Ensure that none of the accessories are selected

    - If you want to do a temperature controlled run, contact the Instrumentation Technician to set up the water bath accessory.

    7. Under the Analyze tab:a. Select the Auto Calculate check box to automatically perform a rate

    calculation on collected data at the end of each run. b. Select Advanced Calculate to set up multiple rate calculations for the

    kinetics run. - Enter the number of different rate calculations that you require in

    the Number of Stages entry field. The number you set here will be reflected in the table below.

    c. Set up the Stage Start and Stop times and select the reaction Order for each of these reaction stages.

    d. If you select a 'first order' or 'second order' Simple Calculate rate calculation you can use the Manual Guess group to manually enter the parameters: A0, AInf and Rate (k). It is presumed that you have a reasonable idea of the values for these fit parameters, as they will be used as a first guess for the Marquardt non-linear regression analysis.

    e. Enter a value in the Factor field to calculate enzyme activity. The numerical multiplication factor is applied to the absorbance.

  • Spectrophotometer Standard Operating Procedure 19

    f. If you are performing a second order reaction, enter the initial concentration of substrate before reaction.

    g. Select Display Fit to automatically overlay the calculated lines of best fit onto the plotted data.

    8. Under the Samples tab:a. Enter the number and names of the samples to be analyzed.

    9. Set up reporting and printing requirements as desired (see section 6.3.1steps 8 and 9, for details).

    10.Finish Setup -- Once you are satisfied with your method setup select OK to confirm any changes you have made and close the Setup dialog. Save the method if you plan to use it regularly.

    6.6.2 Kinetic measurement without using the RX accessory1. Click the Zero button to zero the system. Alternatively, select Zero in the

    Commands menu to perform a zero. A Loading Guide dialog box will be displayed.

    2. If you want, change the name of the blank. 3. Place the blank solution in the sample compartment and click OK. 4. The system will perform an instrument zero on the blank solution. Press the

    Start button to start a data collection. Alternatively you can select Start in the Commands menu. Do not add your active reagent at this time. The system will display the Save As dialog box.

    5. Enter the file name for this kinetics run in the File name field and press Save. The system will display a Loading Guide dialog box.

    6. If you want, change the name of the sample. 7. Place the sample solution in the correct cell position and click OK. The

    system will set up the Graphics area and the Sync Start dialog box will appear.

    8. Add your active reagent just before the Count Down reaches 0:00 or commence the data collection by pressing the OK button.

    6.6.3 Setting up the RX accessory1. See Figure 6-1 for an illustration of the RX accessory2. CAUTION:

    a. The pneumatic drive must never be operated when there is a gap between the syringe plungers and the drive ram or with a gap between the stop syringe plunger and the copper leave trigger.

    b. The drive must not be used to fire a single drive syringe.c. Never thermostat the unit below 1.5 C when using aqueous solution.d. Never connect or disconnect any electrical leads, when the unit is under

    power.e. Do not use HF in any form with the instrument

    3. Flush the system with the blank solution (i.e. pure water)a. Attach the waste reservoir syringe to the valve adjacent to the stopping

    syringeb. Set the reagent control valves ( ) to the loading position

  • Spectrophotometer Standard Operating Procedure 20

    c. Push the drive syringes fully forward.d. Set the exhaust valve to the fully closed position (indicator arrows

    pointing downwards and to the right).e. Fill the reagent syringes with blank solution and attach to the luer lock

    ports on the top of the RX accessory. f. Gently pull the drive syringes back to fill with blank solution (i.e. load the

    drive syringes).g. Set the reagent control valves to the drive position.h. Set the exhaust valve open to the waste reservoir syringe (indicator

    arrows pointing upwards and to the right).i. Push the drive syringes fully forward to flush out the flow lines.j. Repeat the blank flush at least three times.

    4. Flush the stopping syringe and test data collection:a. Set the reagent control valves to the loading position and reload the drive

    syringes with the blank solution.b. Set the exhaust valve to open the stopping syringe to the waste reservoir

    (the indicator arrows should point upwards and to the left).c. Push the stopping syringe piston fully forward.d. Set the exhaust valve to connect the stopping syringe to the main flow

    line (the indicator arrows should point downwards and to the left).e. Press the green button to load the stopping syringe and initiate date

    collection.f. Repeat at least three times to ensure that the system is completely

    flushed and functioning properly.5. Conduct a blank measurement:

    a. Load the drive syringes with the blank solution.b. Click on Zero to zero the detector.c. Click on start; a prompt will appear on the screen with a count down from

    2 minutes to zero. You have to press the green button within this time frame to initiate data acquisition.

    d. Make sure the signal is flat. If it is not, there are bubbles in the line and the blank measurement should be re-done.

    6.6.4 Sample measurement1. Measure a kinetic reaction:

    a. Load one drive syringe with one reactant, and the other with the second reactant, and flush the system as outlined in step 3.

    b. Repeat with the stopping syringe in operation (step 5).c. Click start and press the green button.

  • Spectrophotometer Standard Operating Procedure 21

    Loading Position

    Drive Position

    Flow line closed Flushing: flow line open to waste reservior

    Drive position: flow line open to stopping syringe

    Flushing: flow line open to waste reservior

    Sto

    pp

    ing

    syr

    ing

    eS

    top

    pin

    g s

    yrin

    ge

    Loading Position

    Drive Position

    Flow line closed Flushing: flow line open to waste reservior

    Drive position: flow line open to stopping syringe

    Flushing: flow line open to waste reservior

    Sto

    pp

    ing

    syr

    ing

    eS

    top

    pin

    g s

    yrin

    ge

    Figure 6-1: RX Accessory loading and drive positions

    6.6.5 Cleaning the RX Accessory1. Cleaning is generally performed in three steps.

    a. Using 10 ml reservoir syringes flush the drive syringes with at least 30 ml of distilled water. Fill the drive syringes with fresh distilled water. Turn the control valves to the drive (forwards) position. Make sure that there is no gap between the drive ram and the syringe plungers. Flush the system at least 10 times with distilled water.

    b. Refill the drive syringes with distilled water. Turn the control valves to the drive (forwards) position. Completely empty the stop syringe. Push the drive ram up, by hand, until the stop syringe is full. Repeat at least three times.

    6.7 Concentration

    6.7.1 Setting up the method parameters1. NOTE: A detailed description of all of the set-up parameters is listed on the

    Cary 50 Help and Videos CD.2. Select the Cary Icon from the Desktop and choose Concentration3. Set the method for the analysis: Select File/Open Method from the menu to

    load a previously defined method and skip to Section 6.7.2. If you dont have a method, select the Setup button to display the Setup dialog and specify the method parameters for a new method by following steps 4 through 9.

    4. Under the Cary tab set the instrument parameters as follows:a. In the Wavelength field, enter the wavelength that you want to monitor.b. In the Ave. time field, set the amount of time, in seconds, for which data

    is averaged. Average time is automatically set for 0.1000 seconds but can be adjusted if needed.

  • Spectrophotometer Standard Operating Procedure 22

    c. Select Replicates or Sample/Std Averaging if you desire, and enter the appropriate number.

    d. Enter an upper range and lower range value in the Y min and Y max entry fields to specify the displayed ordinate range. Note that these are starting values only. The Win UV software will automatically rescale the calibration graph as the standards are measured.

    e. Select the Status Display check box so that you can view various instrument parameters during the scan to setup visual system monitoring.

    5. Select the Standards tab to set up the standards and their parameters associated with the data collection.

    a. Select 'Calibrate During Run' to perform a calibration when the Start button is pressed.

    b. Set the appropriate Units for your standards for reporting purposes. c. Set the Standards field to the number of standards that you are using in

    your calibration. The table below this field will expand or contract to match your choice.

    d. In the Standards table, enter the concentration of each standard into the Conc column.

    e. Select the type of curve fitting required for your calibration in the Fit Type group.

    f. Enter the required R2 value or correlation coefficient into the Min R2 field. The closer the number is to 1.000 the better the fit. Typically, 0.95 is used.

    6. Under the Accessories tab, select the accessories to use in the analysis:a. Select the Accessories 1 and Accessories 2 tabs and make sure that no

    accessories are selected.7. Under the Samples tab:

    a. In the Sample Names box:- Fill in the number of samples to be run and enter the sample names.

    b. Set up the weight and volume correction:- Select the Weight/Volume Corrections check box. - Enter the theoretical sample weight in the Method Weight field. This

    is the weight of the sample specified in your method. (The Actual Weight is entered for each sample in the Sample Names list.)

    - Enter the units in the Weight Units field.- Enter the theoretical sample volume in the Method Volume field.

    This is the volume to which the method tells you to make the sample. (The Actual Volume is entered for each sample in the Sample Names list.)

    - Enter the units in the Volume Units field. - In the Sample Names table, enter the Actual Weight and Actual

    Volume for each sample. 8. Set up reporting and printing requirements as desired (see section 6.3.1

    steps 8 and 9 for details).9. Finish Setup -- Once you are satisfied with your method setup select OK to

    confirm any changes you have made and close the Setup dialog. Save the method if you plan to use it regularly.

  • Spectrophotometer Standard Operating Procedure 23

    6.7.2 Sample and standard measurement1. Select the Zero button from the application window, place a blank in the

    sample compartment and select OK.2. Press the Start button or select Start in the Commands menu. The

    Standard/Sample Selection dialog box will appear. 3. Select the standards and samples to be used in the analysis. 4. Click OK to exit the Standard/Sample Selection dialog box. 5. The Present Standard dialog box will prompt you to place the appropriate

    standard in the sample compartment. Press OK to measure the standard. 6. Repeat until you have measured all the standards. The Cary will calculate

    the calibration and the correlation coefficient. a. Note: If the set correlation coefficient (R2) value is not met, the Cary will

    prompt you with 'Min R2 test failed'. When you press OK, the Cary will then prompt you with 'There is no valid calibration. Proceed in Abs (or Emission)?'. If you click Cancel, the Concentration run will finish. If you click Yes, the Cary will measure the absorbance or emission of any presented samples, but will not generate a concentration.

    7. Once all the standards have been read, the Present Sample dialog box will prompt you to place the appropriate sample in the sample compartment. Press OK to measure the sample and calculate its concentration. (If replicates have been nominated, then the concentration is calculated after the final sample replicate is read.)

    8. Repeat for the remaining samples. 9. Save your results as a batch file.

    6.8 RNA/DNA

    6.8.1 Setting up the method parameters1. NOTE: A detailed description of all of the set-up parameters is listed on the

    Cary 50 Help and Videos CD. 2. Select RNA-DNA from the Cary WinUV menu.3. Set the method for the analysis: Select File/Open Method from the menu to

    load a previously defined method and skip to Section 6.8.2. If you dont have a method, select the Setup button to display the Setup dialog and specify the method parameters for a new method by following steps 4 through 10.

    4. Under the Cary tab set the instrument parameters as follows:a. Enter the first and second wavelengths at which you would like to

    measure your sample.b. If you require background correction, select the Background Correction

    check box and enter a background wavelength in the corresponding field. c. If you are performing a wavelength scan, check 'Scan Samples'. The

    Display Options group will be activated and the Baseline tab will appear. d. Enter a Start and Stop wavelength value and select a Scan Rate. e. In the Display Options group, select the way in which you want the data

    displayed as it is collected. Choose the Individual Data radio button to

  • Spectrophotometer Standard Operating Procedure 24

    display the collected data of each sample in individual graph boxes. Choose the Overlay Data radio button to superimpose the collected data of each sample in the run in one graph box.

    5. Select the Baseline tab and check 'Baseline correction'. To use a previously stored baseline (*.CDN), click on the Baseline button and browse for the appropriate file. Otherwise, you can perform your baseline correction at the beginning of the run.

    a. Select the Status Display check box so that you can view various instrument parameters during the scan to setup visual system monitoring.

    6. Under the Accessories tab, select the accessories to use in the analysis:a. Ensure that none of the accessories are selected

    - If you want to do a temperature controlled run, contact the Instrumentation Technician to set up the water bath accessory.

    7. Enter a list of sample names under the Samples tab: a. Enter the number of samples that you are going to use in the Number of

    Samples field. The Sample Names list below will expand or contract to match your choice.

    b. In the Sample Names list, enter the name of each sample. You can enter up to 20 characters for each name.

    c. De-select any samples that you don not wish to analyze by clicking beside the desired sample in the small first column to remove the red tick.

    d. If the samples have the same name with a different numeric extension, enter the name in the first sample position and then press the Increment button.

    e. If you would like multiple readings of the same aliquot, select the Replicates check box and enter the number of replicates required in the data entry field that appears.

    8. Set up analysis parameters under the Analyze tab:a. If you would like to calculate any Warburg Christian or 260 nm Factor

    parameters, make your selections here.9. Set up reporting and printing requirements as desired (see section 6.3.1

    steps 8 and 9 for details).10.Finish Setup -- Once you are satisfied with your method setup select OK to

    confirm any changes you have made and close the Setup dialog. Save the method if you plan to use it regularly.

    6.8.2 Sample Measurement1. Zero the instrument.

    a. Place the blank solution or empty cuvette in the sample compartment of the Cary Eclipse instrument. Make sure not to touch the side of the cuvette while doing so.

    b. Click the Zero button to zero the system. Alternatively, select Zero from the Commands menu to perform a zero.

    c. When the result is zeroed, the word 'Zeroed' will appear in the Y display box in the top left corner of the Scan Application window.

  • Spectrophotometer Standard Operating Procedure 25

    2. Collect baselines: If you have selected to perform a baseline correction and are not using a stored baseline, take a baseline reading now by following the steps below. Otherwise, proceed to step 3.

    a. Press the Baseline button to collect a baseline for each cell. A Cell Loading Guide dialog box will appear.

    b. Load the blank/s as depicted. c. Click OK. On completion of the baseline collections, the word 'baseline'

    will appear in red above the ordinate instrument status reading. 3. Insert sample into the sample holder. 4. Select the Start button to commence a data collection. Alternatively, select

    Start from the Commands menu. The Sample Name dialog is displayed. Place the sample in the sample compartment of the Cary Eclipse instrument.

    5. In the Sample Name dialog, enter the appropriate name for you sample and select OK. The scan will commence and the trace will appear in the Graphics area.

    6. Save the collected data -- Once the run is finished, select the Save As command from the File menu. In the File name field, enter a file name for this scan run.

    6.9 Thermal Not currently available on the Cary 50

    6.10 Enzyme Kinetics

    6.10.1 Setting up the method parameters1. NOTE: A detailed description of all of the set-up parameters is listed on the

    Cary 50 Help and Videos CD.2. Select Enzyme Kinetics. 3. Set the method for the analysis: Select File/Open Method from the menu to

    load a previously defined method and skip to section 6.10.2. If you dont have a method, select the Setup button to display the Setup dialog and specify the method parameters for a new method by following steps 4 through 10.

    4. Select the Cary tab and specify the Instrument parameters for your analysis. a. Enter the Wavelength and Ave Time you require in the corresponding

    entry fields. b. Enter an upper range and lower range value in the Y min and Y max entry

    fields to specify the displayed ordinate range. c. Select the Advanced Collect radio button in the Collect Timing group. This

    enables you to set up different data collection procedures for the multiple rates in your reaction.

    d. Enter the number of different reaction rates that you require in the Number of Stages entry field. The number you set here will be reflected in the table below.

    e. Specify how long the Cary will wait after reading each cell before it starts another reading cycle by setting the Cycle time for each rate Stage.

  • Spectrophotometer Standard Operating Procedure 26

    f. Specify the duration of the measurement run by setting the Stop time for each rate Stage.

    5. Under the Options tab, select the way in which you want the data displayed as it is collected.

    a. Choose the Individual Data radio button to display the collected data of each sample in individual graph boxes. Choose the Overlay Data radio button to superimpose the collected data of each sample in the run in one graph box.

    6. Under the Accessories tab, select the accessories to use in the analysis:a. Ensure that none of the accessories are selected

    - If you want to do a temperature controlled run, contact the Instrumentation Technician to set up the water bath accessory.

    7. Select the Analyze tab. a. Set up the Start and Stop times for your V0 Calculation. b. Enter the correct product absorptivity for your reaction. c. Enter the correct cell pathlength for your reaction. d. Select the method by which the data obtained from your selection in the

    Plot/Fit group will be analyzed. Choose Linear Least Square or Marquardt by selecting the appropriate radio button.

    e. Choose the inhibitor model for your analysis. Select either the Non Competitive, Competitive or Uncompetitive radio button.

    f. Check the box/es beside the Plot/Fit type/s that will be used to determine Vmax and Km values.

    g. Select the Auto Calculate check box to automatically perform enzyme kinetics calculations on collected data at the end of each run. These results will be displayed in the Report area.

    8. Select the Samples tab:a. Enter the number and names of the samples to be analyzed.b. Enter the sample and inhibitor concentrations for each sample.

    9. Set up reporting and printing requirements as desired (see section 6.3.1steps 8 and 9 for details).

    10.Finish Setup -- Once you are satisfied with your method setup select OK to confirm any changes you have made and close the Setup dialog. Save the method if you plan to use it regularly.

    6.10.2 Sample measurement1. Click the Zero button to zero the system. Alternatively, select Zero in the

    Commands menu to perform a zero. A Cell Loading Guide dialog box will be displayed.

    2. If you want, change the names of the blank samples. a. Place the blank solution in the cell and click OK.

    3. Press the Start button to start a data collection. Alternatively you can select Start in the Commands menu. Do not add your active reagent at this time. The system will display a Cell Loading Guide dialog box.

    4. If you want, change the names of the samples.

  • Spectrophotometer Standard Operating Procedure 27

    5. Place the sample in the cell and click OK. The system will set up the Graphics area and then display the Save File dialog box.

    6. Enter the file name for this run in the File name field and press Save.The Sync Start dialog box will appear.

    7. Add your active reagent just before the Count Down reaches 0:00 or commence the data collection by pressing the OK button.

    8. Once the run has started, enter the substrate and inhibitor concentrations if not entered previously on the Samples tab:

    a. Open the User Data Form, by right clicking in a graph box and selecting User Data Form from the list or by selecting it from the Graph menu.

    b. The table that appears has Data Names and may have V0 values already entered in the first two columns. In the third and fourth columns, enter your values for [S] and [I] in the appropriate units.

    c. Press OK. Your [S] and [I] values are now ready to be used in calculations, and the Cary will perform the calculations at the end of the run.

    9. Repeat for the remaining samples. 10.Save your results.

    6.11 Scanning Kinetics

    6.11.1 Setting up the method parameters1. NOTE: A detailed description of all of the set-up parameters is listed on the

    Cary 50 Help and Videos CD.2. Select Advanced Reads. 3. Set the method for the analysis: Select File/Open Method from the menu to

    load a previously defined method and skip to section 6.11.2. If you dont have a method, select the Setup button to display the Setup dialog and specify the method parameters for a new method by following steps 4 through 9.

    4. Under the Cary tab set the instrument parameters as follows:a. Set the wavelength range for the scan by entering the values you require

    in the Start/Stop fields, for example 500/400. b. Enter an upper range and lower range value in the Y min and Y max entry

    fields to specify the displayed ordinate range. c. You now need to set the speed of the data collection. With the Cary

    instruments you do this by setting the Ave Time and Data Interval: - In the Ave Time field enter the required value. 0.1 sec is a good

    starting value. - In the Data Interval field, enter the wavelength increment you

    require between data points. 0.5 nm is a good starting point. The Cary will automatically update the Scan Rate field when you select it.

    d. Select the Advanced Collect radio button in the Collect Timing group. This enables you to set up different data collection procedures for the multiple rates in your reaction.

  • Spectrophotometer Standard Operating Procedure 28

    - Enter the number of different reaction rates that you require in the Number of Stages entry field. The number you set here will be reflected in the table below.

    - Specify how long the Cary will wait after reading each cell before it starts another reading cycle by setting the Cycle time for each rate Stage.

    - Specify the duration of the scanning kinetics run by setting the Stop time for each rate Stage.

    5. Select the Show Status Display check box on to display various information fields on your current reaction.

    6. Select the Baseline tab. a. Select the Baseline correction radio button. This will force the Cary to use

    a baseline scan to perform a baseline correction on the sample data. The correction will be performed on each point before it is displayed.

    b. Under the Accessories tab make sure that no accessories are selected.7. Note: the Analyze tab is used for post-run analysis. 8. Set up reporting and printing requirements as desired (see section 6.3.1

    steps 8 and 9 for details).9. Finish Setup -- Once you are satisfied with your method setup select OK to

    confirm any changes you have made and close the Setup dialog. Save the method if you plan to use it regularly.

    6.11.2 Sample measurement1. If you do not have a valid baseline file, the Cary will prompt you to click the

    Baseline button. Click the Baseline button to set up the baseline collection. a. If you want, change the name of the blank. b. Insert blank samples into the cell changer to collect the 0Abs/100%T

    baseline scans and click OK. c. The system will set up the Graphics area and the Cary will collect the

    baseline scan. After the collection, the word 'baseline' will appear in red in the ordinate status box, indicating that you are in baseline correction mode and you have a valid baseline file for the correction.

    2. Click the Zero button to zero the system. Alternatively, select Zero in the Commands menu to perform a zero.

    a. If you want, change the name of the blank. b. Insert blank sample into cell holder and click OK.

    3. Click the Start button to start a data collection. Alternatively you can select Start in the Commands menu. Do not add your active reagent at this time.

    a. At this point, the system will display the Save File dialog box if you have selected Storage On (Prompt at Start) on the Auto Store tab of the Setup dialog. If so, enter the file name for this Scanning Kinetics run in the File name field and press Save.

    4. The system will display a Cell Loading Guide dialog box. If you want, change the names of the samples.

    5. Place the sample solution(s) in the correct cell positions and click OK. The Sync Start dialog box will appear.

  • Spectrophotometer Standard Operating Procedure 29

    6. Add your active reagent just before the Count Down reaches 0:00 or commence the data collection by pressing the OK button.

    a. At the end of the run, determine the actual Stop Time by observing the last value in the Time column of the User Data Form.

    7. Repeat for the remaining samples. 8. Save your results.

    6.12 General AnalysisOnce a plot has been drawn, you can use several buttons on the toolbar to manipulate the graph.1. Free mode: the cursor (which appears as a +) can be moved in any direction

    without any restrictions.2. Track mode: along with the cursor, a set of intersecting lines will appear.

    As you drag the cursor to the left or right, the horizontal line rides along the line produced by the data points. You can monitor the X and Y values that result from specific data points by looking in the right-hand corner beneath the graph.

    3. Track Preferences: This displays the names of the lines generated from your data points and their corresponding colors and filenames.

    4. Graph Preferences: This allows you to change the color and width of the axes, as well as the font and the way the data is plotted (i.e. dots or solid lines).

    5. Scale Graph: This allows you to change the scale of the graph by typing in the area you would like to focus on.

    6. Add Label: This feature allows you to add labels to your graph.7. Sometimes no peaks will appear. This may be due to the peak threshold. To

    adjust the peak threshold, go to Graph Peak Labels Threshold and adjust the threshold accordingly.

    6.13 Instrument Shut-down1. Fill in the relevant details in the log book.2. Close the Cary WinUV application and shutdown Windows (if you do not

    close the Cary software, the lamp will remain on and this will significantly decrease the life of the lamp).

    3. After Windows has shutdown completely, HOLD DOWN the power button on the computer for 8-10 seconds to shutdown the computer and the instrument.

  • Spectrophotometer Standard Operating Procedure 30

    7. QUALITY CONTROL

    7.1 Instrument ValidationThe Validate Application can be used to optimize the settings and validate the accuracy of the Cary Eclipse Instrument by executing a number of pre-defined tests. The tests are preset with default parameters that comply with international standards for Good Laboratory Practices. This application can be used to perform wavelength checks and calibrations of various Cary Eclipse functions.

    The following validation tests are run once every three months by the Instrumentation Technician:1) Wavelength Accuracy Test / Xenon Line Scan Test:

    Poor wavelength accuracy can produce low quantitative results as the intensity measurement may have been made on the side of the peak rather than at the peak. Wavelength accuracy is determined by scanning a known wavelength peak and calculating the difference between the theoretical wavelength peak and the wavelength peak as reported by the instrument.

    2) Photometric Noise Test:The noise level is an indication of the stability of a reading. It determines the precision of the measurement and the detection limits of the instrument. this test measures the photometric noise over a two minute time frame at various absorbance settings. (This is specified as Root Mean Square (RMS), which is approximately one fifth of the peak to peak value).

    3) Baseline Flatness Test:This test is a measure of the flatness of the signal over the wavelength range. If selected, it will test the baseline flatness using a wavelength scan.

    7.2 Lamp Alignment and CalibrationAll alignment, lamp replacement, and calibration procedures should only be carried out by the Instrumentation Technician.

    For the Cary 50 instrument to operate at peak performance, the lamps must be correctly aligned in the optical path. Poor lamp alignment or the use of old, failing lamps may decrease the signal-to-noise ratio and adversely affect the performance of the instrument. The Align application can be used to perform alignment procedures when the Validate application indicates a problem with the lamp, or after the lamp has been replaced. The xenon lamp has a finite lifetime and must be changed on a regular basis. When the data is becoming noisy or a lamp failure error appears, the lamp may need to be replaced. Note: The Cary 50 Xenon lamp is pre-aligned; only the source mirror requires alignment. The EHT and wavelength calibration can also be carried out if the Validate application indicates a problem.

  • Spectrophotometer Standard Operating Procedure 31

    8. TROUBLESHOOTINGTroubleshooting tips are from the Cary Help and Videos CD.

    8.1 General

    Q. Is there some way I can check my Windows setup for the Carysoftware?

    A. Yes, in the Cary WinUV folder and Start menu entry, there is a 'Troubleshoot Windows' option. Select this and follow the instructions on the screen to solve problems with running the software and online Help. (This wizard is only applicable to Windows 95 and NT4 users.)

    Q. I cannot see an OK or Cancel button on a dialog so I cannot set up my method. What do I do?

    A. You have your screen resolution set too low. Right click on the Windows Desktop, select Properties and click on the Settings tab. Change the Desktop Area setting to 800 x 600. Make sure that the Color Palette is set to High colour (16 bit).

    Q. I cannot find how to set the correct operating (abscissa) mode for my run. How do I set this?

    A. Check that you are using the correct application. For example, time-based measurements are performed in the various kinetics applications.

    Q. How do I generate a report from an existing file?

    A. Press the Recalculate button and make changes as required in the dialog that appears. When you have finished, use the Print button or the Print command in the File menu to print your new report. You can view the report on screen before printing by using the Print Preview command in the File menu.

    Q. I want to export my data to a spreadsheet file. When I press Recalculate, the Autoconvert options are grayed out. How do I do this?

    A. You need to use the Save Data As option in the File menu. In the dialog that appears, set the Files of type field to Spreadsheet ASCII (csv) and your data will be exported. You can also refer to 'How to export collected data' in your application's Help.

    Q. I have saved my sample file, but now I cannot find it in the directory. What has happened?

    A. You may have saved the file as a different file type, for example as a Datafile rather than a Batch file. To see all the files in the directory, click Open Data in the File menu and set the Files of type field to All Files.

    Q. How do I include details of the method when I export data to a spreadsheet file?

    A. Select the option Select for ASCII (csv) with Log in the Autoconvert group on the Reports tab of the Setup dialog.

  • Spectrophotometer Standard Operating Procedure 32

    Q. The Cary software is running slowly, how do I get it back to normal?

    A. You may have too many traces available in the Trace Preferences dialog. Delete any unwanted traces, and the speed of your application will increase. See 'How to remove a trace from the trace list' in your application's Help.

    Q. I have typed in an ADL program, but it is not carried out when I press the Start button. How can I apply my ADL program to a data collection?

    A. You need to use the User Collect option that is available in most applications on the Cary tab of the Setup dialog. Enter your ADL commands in the User Result line that appears when you activate User Collect.

    Q. I need to store information about a trace and print it in my report e.g. sample preparation details such as pH or the reagents used. How do I do this?

    A. Use the User Data Form feature. This can be accessed from the Graphics menu or from the toolbar.

    8.2 Starting a runQ. I have a 'Connect' button instead of a 'Start' button. What does this

    mean?A. Only one Cary application can communicate with the instrument at a

    time. You have another Cary application open that is online with the Cary. Provided that the other application is not collecting data, you can press Connect to make this application go online and the Start button appear.

    Q. I only have one application running, yet I am still seeing a Connect button instead of a Start button. Why?

    A. The Cary instrument is still initializing and the System Information application has control over it. When the initialization has finished, your chosen application will have control and the Start button will become active.

    Q. I have set up a method and want to press Start, but the Start button is grayed out. Why is this?

    A. You may be using a thermostattable accessory, and the Cary is waiting for this to reach the required temperature before it allows you to commence the run.

    Q. Why do I sometimes get asked twice for a sample name when I press Start?

    A. Storage on (Prompt at start) may be selected in the Autostore tab in the Setup dialog. The first prompt by the Cary is for the batch file name into which your data will be stored. The second prompt is for the name of the trace within the batch file. This appears in the Trace preferences dialog

  • Spectrophotometer Standard Operating Procedure 33

    and in your report. You cannot change this name once it is entered.Q. I need to set up the Cary to perform a simple calculation during the

    run. How do I do this?A. You need to use the User Collect option that is available in most

    applications on the Cary tab of the Setup dialog. Enter your ADL commands in the User Result line that appears when you activate User Collect.

    Q. I use the same User Collect statement in a number of different methods. Can I store this?

    A. Yes, you can store User Collect statements on the Edit tab of the System Information application.

    Q. How do I store information about a trace and print it in my report e.g. sample preparation details such as pH or the reagents used?

    A. Use the User data form feature. This can be accessed from the Graphics menu or from the toolbar.

    8.3 Data collectionQ. My kinetics run stops before the last data point is measured, why?

    A. You have the Stop time on the Cary tab of the Setup dialog set too short. Increase the time so that the instrument has enough time to complete all the cycles you have specified.

    8.4 Graphics

    8.4.1 General

    Q. I accidentally deleted all my traces from my graph box by pressing Clear All Traces. Can I get them back?

    A. Yes, open the Trace preferences dialog (via the Graph menu or the toolbar button) and then mark the traces you require in your graph as Visible.

    Q. I can find a Zoom Out button, but not a Zoom In button. How do I enlarge an area of the screen?

    A. You need to use the Free Cursor (accessed from the Cursor mode option

    in the Graph menu or by clicking the toolbar button) and then click and hold down the left mouse to create a box around the area of interest. See 'How to zoom a graph' in your application's Help.

    Q. Why does choosing to display my traces in different line formats have no effect?

  • Spectrophotometer Standard Operating Procedure 34

    A. The different line formats will only alternate when the Trace width has been selected at 1 pixel. Style cycling and Trace Widths are selected in the Graph preferences dialog accessed from the Graph menu or by

    clicking the toolbar button.Q. When I autoscale a graph with several traces in it some of the traces

    are drawn off the edge of the graph box. How do I autoscale them all?A. The autoscale function uses the currently focused trace to autoscale. Click

    on the trace which has the most variation in terms of Y-scale and then use the Autoscale function.

    Q. It is frustrating to set up axes colors and widths etc in the Graph Preferences dialog box, only to find that next time the instrument is turned on they are lost.Is there a way to save these settings?

    A. To store the Settings from the Graph preferences dialog, such as axes color, and width, so that the system remembers these settings every time an application is opened you will need to do the following:

    1. Open the application of interest 2. Add a graph to the graphics area 3. Open the graph preferences dialog and setup all your graph settings 4. From the file menu select the Save Data As menu and save your

    settings as a batch file. For example 'Kinetics Default settings.bkn'. 5. Create a shortcut on the desktop for this batch file. 6. Next time you want to open this application, rather than clicking on

    the icon in the Cary WinUV folder on the desktop, click on the shortcut (e.g. 'Kinetics Default settings.bkn') icon instead and you will get your graph preferences load automatically.

    8.4.2 Graph Labeling

    Q. Why can't I add a label to my graph?

    A. You may not have selected the graph box. You need to first click on the graph box you require to select it. A black line with eight 'handles' will appear around the box. There must also be at least one trace Visible in the graph box before you can add a label to it.

    Q. How can I store information with my graph so I can print it every time I run a sample? i.e. I do not want to add labels to every sample trace.

    A. The solution is to add the sample information to the graphics background and then save this as a graphics template. See 'How to add a label to a graph' in your application's Help.

    Q. When I use the pea