Buffer Preparation Guide. School experiments. Tips and Hints. for ph Measurement and learn Pipetting easily

Transcription

1 Buffer Preparation Guide School experiments Tips and Hints A Natural Guide science to Biological laws experience Buffer Preparation live for ph Measurement and learn Pipetting easily

2 2Introduction

3 Dear Reader, Researchers in Life Sciences carry out work on biological molecules, such as antibodies or proteins, in order to modify them for their purposes, often in the search for disease cures and prevention. Almost all biological processes are ph dependent. The purpose of a buffer in a biological system is to maintain intracellular and extracellular ph within a very narrow range and resist changes in ph in the presence of internal and external influences. The rather complex laboratory buffer preparation process involves a number of different steps, including amount calculation, substance weighing, ph solution value control and liquid pipetting. Over the years, METTLER TOLEDO has gained a deep understanding of ph determination and liquid pipetting and we have developed this buffer preparation guide, containing useful tips and hints for the use of pipettes and ph Meters, in order to pass on some of this knowledge. Yvonne Appoldt Marketing Manager LAB Division Marketing Urs Alig Segment Marketing Specialist LAB Division Marketing 3

4 4Buffer Concept 1. The Buffer Concept 1.1 Buffer Definition Buffers are aqueous systems that resist changes in ph when small amounts of acid or base are added and are composed of a weak acid and its conjugate base. A buffer keeps the ph of a solution constant by asorbing protons that are released during reactions or by releasing protons when they are consumed by reactions. The discovery that partially neutralized solutions of weak acids or bases are resistant to changes in ph, when small amounts of strong acids or bases are added, led to the concept of the buffer. =O =O CH 3 C OH CH 3 C O +H + weak acid Predominates Negligible Fig. 1: Most buffers are weak acids and their salts dissociate in a solution as shown 1.2 Biological Buffers Different inorganic substances were originally used as buffers (e.g. phosphate, cacodylate, borate and bicarbonate) and later, weak organic acids were also used. Many of these buffer substances, however, have the disadvantage of not being inert and have lasting effects on the system under investigation (e.g. inhibition of enzymes or interactions with enzyme substrates etc.). Biochemical reactions are especially sensitive to ph. In all multi-cellular organisms, the fluid within the cell and the fluids surrounding the cells have a characteristic and nearly constant ph. The purpose of a biological buffer in a biological system, therefore, is to maintain the intracellular and extracellular ph within a very narrow range and resist changes in ph in the presence of internal and external influences.

5 1.3 General Requirements of Biological Buffers Biological buffers used in cell cultures, isolation of cells, enzyme assays and other biological applications must possess the following distinctive characteristics. 1. Solubility The buffer should be freely soluble in water and poorly soluble in other solvents. 2. Permeability through biological membranes The buffer should not be able to permeate biological membranes to prevent concentration in the cell or organelles. 3. Ionic strength The buffer should not alter the ionic strength of the system. 4. Dependence of pk a value The pk a value of a buffer should be influenced as little as possible by the buffer concentration, the temperature and the ion composition of the medium. 5. Inert substances The buffer should not be subject to either enzymatic or non-enzymatic changes, i.e. it should not be an enzyme substrate or enzyme inhibitor and should not react with metabolites or other components. The buffer should, therefore, be inert. 6. UV absorption Buffers should not absorb any light at wave-lengths longer than 230nm, since many spectrophotometric investigations are performed in this range (determination of DNA, RNA and protein concentrations). 5

6 Laboratory Solutions 2. Buffer Preparation Process and METTLER TOLEDO Solutions The buffer preparation procedure consists of around 8 process steps, including differential weighing, pipetting and ph determination processes. METTLER TOLEDO solutions make this procedure easy and reliable with the help of: XP Precision Balances LabX laboratory software S20 Seven Easy ph Meters Rainin Pipet-Lite or Rainin AutoRep pipettes Label printers Step 1: Calibration Calibrate instruments easily METTLER TOLEDO offers simple and reliable routine testing solutions for balances, ph meters and pipettes. CarePacs test weights for smooth routine balance testing. Chemical buffers for exact meter ph calibration. Evaporation traps for occasional pipette performance tests. Step 2: Calculation Step 3: Weighing-in Calculate the amount of chemicals to weigh in LabX software provides full step-by-step SOP user guidance on the balance screen. Calculations and documentation are completed automatically ensuring secure results. } Weigh in chemicals securely with XP Precision Balances Brilliant colors and high-resolution graphics guide the user through the weighing application. The graphical SmartTrac guide provides a quick overview of target weight and tolerances for the fast and exact weighing in of chemicals. } 6

7 Step 4: Addition of solvents Add solvent quickly with Rainin AutoRep Pipette The AutoRep pipette saves time and speeds up long pipetting series whilst remaining fatigue-free and precise. The flexible volumes and wide selection of easy-to-exchange tips ensure optimal performance on any given volume from 1μl 50ml. } Step 5: Dissolving substances Dissolve substances easily Use the Magnetic Protection Shield Weighing Pan (MPS) on the XP Precision Balance when using magnetic stir bars to dissolve chemicals. The MPS securely eliminates adverse weighing influences caused by the magnetic stir bars. } Step 6: ph adjustment Check and adjust ph value correctly The S20 SevenEasy ph Meter, with its intuitive interface and visual icons, makes ph measurement simple even for untrained users. With the addition of the InLab Routine Pro ph Electrode, with its integrated temperature probe for correct automatic temperature compensation, checking and adjusting ph becomes a quick and accurate task. } } Step 7: Buffer volume adjustment Adjust buffer to final volume with precise pipetting The Rainin Pipet-Lite is the most ergonomic, precise and durable manual pipette on the market for routine pipetting avoiding tired hands and repetitive strain injuries. } Step 8: Label generation Generate labels automatically LabX Software automatically stores buffer results and prints labels for buffer solutions. This ensures safe data transfer and clearly readable labels. } 7

8 8pH Measurement 3. ph Measurement Tips and Hints 3.1 Tips and Hints for Buffer Preparation Exact and reliable ph Measurement is important for the correct preparation of buffers. Here are some useful tips and hints for ph determination when preparing buffers. 1. Use a ph electrode with integrated temperature probe Combined electrodes (figure 2) with an inner ph sensor and an outer reference element are very commonly used. They house a temperature sensor in the same body as the ph and reference elements in order to further simplify ph measurements. This allows temperature compensated measurements to be made. Screw Cap, S7 or MultiPin head Refill opening, SafeLock Reference electolyte METTLER TOLEDO InLab Routine Fig. 2: Combined electrode with integrated temperature probe Ceramic junction Silver-ion Trap ARGENTHAL reference system Integrated temperature probe ph-sensitive glass membrane 2. Immerse electrode no longer than necessary It is important that the ph electrode is not left immersed in solution any longer than necessary especially when using a solution that contains proteins. After several ph measurements of solutions containing proteins, rinse the electrode in a mild alkali solution and then wash several times with de-ionized water. 3. Clean protein contaminated electrode with a pepsin/hci solution Junctions contaminated with proteins can often be cleaned by immersing the electrode into a pepsin/hci (5% pepsin in 0.1mol/L HCl) solution for several hours. 4. Prevent any bacterial or fungal growth Autoclave or sterile-filter the buffer solution in order to prevent any bacterial or fungal growth. This is important when large quantities of buffers are prepared and stored over a long period of time.

9 3.2 Tips and Hints for ph Determination Ensure that you always measure the correct ph value by following the tips and hints below. ph [H + ] (M) Fig. 3: ph scale Acidic Neutral Basic 1. Warm-up ph Meter A ph meter may require a warm up time of several minutes. When a ph meter is routinely used in the laboratory, it is better to leave it ON with the function switch set at standby. 2. Clean electrode before beginning Prior to beginning; make sure the electrode is well rinsed with deionized water. To clean the electrode, rinse it with de-ionized water after each measurement but never wipe it clean with a tissue. 3. Calibrate with at least two standard buffer solutions 4. Get rid of bubbles inside the electrode Bubbles on the inside of the electrode will make measurements unstable. To get rid of any bubbles, gently shake the electrode with a vertical motion, such as with a fever thermometer. 5. Maintain the identical stirring conditions when calibrating and measuring Use the same stirring conditions when calibrating and measuring, i.e., if no stirring takes place during calibration, do not stir during measurements. 6. Maintain ph electrode regularly to prolonging its lifetime 7. Use the TroubleShooter if performance problems occur A correctly selected ph electrode that has been working properly may, nevertheless, suddenly start performing badly. The ph TroubleShooter gives a detailed electrode diagnosis, as well as tips and hints on how to resolve problems. ph troubleshooter: } 9

10 Pipetting 4. Pipetting Tips and Hints 4.1 General Tips for ph Titration The following tips help to ensure that buffer ph is properly adjusted. 1. Use Precision Pipettes instead of glass transfer Pasteur pipettes The addition of concentrated acids and bases to a buffer in order to achieve the correct ph is often associated with some guesswork, especially for first time buffer preparers. Traditionally, glass transfer Pasteur pipettes have been popular for titration but these do not offer precise knowledge of the volume added. Therefore, we recommend the use of precision pipettes to aid the development of reproducible titration protocols. Fig. 4: EDP3 Electronic Single Pipette from RAININ 2. Use electronic pipettes for reproducible titration protocols Aspiration and dispensing are initiated on electronic pipettes by pressing a trigger rather than using a plunger. By using an electronic pipette, users can improve sample pick-up and dispensing consistency, which, in turn, eliminates most user-to-user technique variability and improves repeatability. Electronic pipettes, such as the Rainin EDP3 (figure 4), often have a Titrate mode, which shows the user precisely how much acid/base was added to a buffer during titration. This speeds up development of reproducible titration protocols. 3. Follow general guideline for ph adjustments As a general guideline, it is safest to start titration very slowly, by adding a volume of acid/base <0.01% of your total buffer volume. Note the rate of ph change and adjust your titration accordingly. Another option is to use a repeater pipette, which combines the advantages of positive displacement, speed and precision. Consider using a large volume pipette or serological pipette if larger volumes need to be added (>1ml) to get any change in ph. When very close to target ph, slow down the addition of reagent Make notes for future buffers Make a note of the volume of acid or base being added during each buffer titration in order to perform the same operation much faster in the future. Sometimes, the correct ph is reached with acid/base remaining in the pipette tip. In this case, it can be difficult to estimate how much acid/base was added to the buffer and we recommend using the titration function on an electronic pipette which keeps track of the dispatched volume displaying very precise information concerning how much reagent was used during titration.

11 4.2 Tips and Hints for Precise Pipetting Pipettes are used in buffer preparation when adding water and additional liquid chemicals to the solution. The tips and hints below explain proper pipetting techniques when preparing buffers. 1. Choose the right pipette For greatest accuracy, work within a pipette range which is 35% of the nominal volume or higher. 2. Keep immersion angle vertical Keep the immersion angle as close as possible to the vertical otherwise the vertical liquid column becomes smaller and too much sample will be aspirated. 3. Use the right immersion depth If the tip is immersed too far, more liquid will be aspirated due to increased pressure. Liquid retained on the tip surface can also distort results. If the tip is not immersed far enough, air can be drawn in resulting in air bubbles and inaccurate volume. 4. Use the correct dispensing technique For most applications, it is recommended to dispense with the end of the tip resting against the vessel wall. This reduces or prevents sample remaining in the tip after dispensing. Remove the pipette by sliding the tip end up the side-wall in order to release any remaining droplet inside the tip. 5. Use the reverse pipetting mode for viscous liquids For dense and viscous liquids, such as glycerol or Tween 20 use the reverse pipetting mode or a positive-displacement pipette. Before sample pick-up, the plunger is pressed completely down to the blowout position. The selected volume of a liquid plus an excess is aspirated into the pipette tip. To dispense, the plunger is pressed only down to the zero position. 11

12 Enhance Daily Research with useful Tips and Tricks Discover more lab solutions for life science research from METTLER TOLEDO on our Academia Internet page. Download useful information, such as technical reports, practical examples, application brochures and guides providing active support for daily research work allowing beginners and professionals alike to benefit from our life science solutions and know-how. } Regular Service Daily research activities will not be interrupted due to inaccurate measurements or broken instruments with our ServiceXXL approach. The value of your investment into METTLER TOLEDO s innovative products deserves to be preserved. Our ServiceXXL approach is a unique blend of service offerings designed to suit your specific business needs, including: Preventive maintenance Extended warranty Calibration Professional documentation } Mettler-Toledo AG CH-8606 Greifensee, Switzerland Phone For more information Subject to technical changes 12/2010 Mettler-Toledo AG Printed in Switzerland Global MarCom Switzerland