Labaratuvar Cam Malzemeleri

Hacim  Liquid volumes are measured in Liters (L), milliliters (mL) or microliters (L)  A liter is slightly more than a quart  A microliter is 1/1000 of a mL or smaller than a tiny drop  Depending on the volume to be measured, 3 options: graduated cylinder, pipet or micropipet

Skala  Bigger units left  Smaller units right  Move decimal point to the right if converting big units to small units  Move decimal point to the left is converting small units to big units

 1.25 L is equal to how many mLs? 1250 mL  How many liters is 60 mLs? 0.06 L  250 uL is how many L? L

Terminoloji  Graduations: lines marked on volume measuring devices that indicate volume  Meniscus: a curve formed by the surface of liquids confined in a narrow space, such as in a measuring device

Terminoloji  TC: To Contain; will contain the specific amount when filled to the capacity mark. It will not deliver the amount if the liquid is poured out because some of the liquid will adhere to the sides of the container.

 TD: To Deliver; marked so that it will deliver the specified amount, assuming the liquid is 20C and is poured out.  Note: plastic containers are considered non-wetting which means water does not stick to them so there is no difference between TD and TC

 Tolerance: how much error is allowed in the calibration or a measuring device.  Volumetric: the most accurately calibrated glassware

Pipetleme  Determine the proper pipette size  Determine the proper pipette roller or bulb Blue: 1 mL pipette Green: 5 or 10 mL pipette  Draw up past your required volume and dispense to your exact fill volume required  Pay attention to the meniscus

 Never mouth pipette  Hold the bottom of the roller and the top of the pipette when you are assembling them  Always keep the pipette vertical when there is fluid in it  Always keep the tip of the pipette sterile and take care to not touch surfaces

 See your hand out for lab practical using disposable pipettes  Pipets with a TD on them indicate they are designed “To Deliver” and that the tiny amount left in the bottom of the pipet after dispensing should NOT be blown out.

 Most QC and R&D laboratories measure very small volumes.  Micropipets are used to measure microliter quantities. What is the symbol for microliter? L  Micropipets are expensive instruments which must be handled carefully

 We have 2 types of pipets in our lab P20: for 2 to 20 L P200: for 20 to 200 L  Let’s take a look at your micropipet diagram handout. Keep this handout out for the micropipet lab exercise

Getting to Know your micropipet  There are 4 parts to a micropipet Plunger button Ejector button Volume display (setter) Dispensing Tip

Getting to Know your micropipet  Plunger button Typically there are 2 stops  The first evacuates the air in the micropipet  The second stop evacuates the volume plus another 50% or so.  Practice to feel the difference

Picking the proper size micropipet  Look at your plunger It will give you the min. and max. that it can deliver  3 numbers will be displayed The top # is the digit for the maximum I.e. 2 is the top # on the P200

Interpret the following settings

Setting your required volume  To set your volume you need to turn the adjusting knob. Rotate slowly to the desired volume setting At no time should the knob be turned past its upper or lower limits. Do not force the settings, the knob will turn easily

Sources of Error for micropipet  Bad pipet tip: leaks, clogged tip  Damaged pipetor  Wrong size tip for pipet size  Bubbles in your tip due to poor technique  Liquid not at room temperature or it may be viscous

Multi channel pipets  In order to increase efficiency, pipets have been designed to hold 4-16 channels at a single dispense.  Most commonly used when running ELISA assays 8 channel pipet

Multichanell Pipettes

Electronic or Automated pipets  Refer to your handout for a picture of an electric pipet. Typically they are battery operated Press a button to pick up, press button to dispense Use to increase or decrease volume settings For large volume labs, automated pipetting instruments are used. Multi channel heads

Burettes  Long graduated tube with a stop cock at one end which is used to dispense known volumes accurately.  See your handout for a picture. There was one out during the scavenger hunt

Calibration  Definition: adjustment of dispensing devices so they dispense accurate volumes.  In the case of pipets we are checking the accuracy and precision of the pipet.

 Accuracy: to deliver the exact quantity required Mean measured volume divided by Nominal or required volume  Precision: the ability to deliver that exact quantity time after time The amount of variation between the each volume measured  1.01, 1.01, 1.02

Calibration Proceduru  Pre Calibration Activities Be sure the Micropipetter is clean Be sure the balance is calibrated and working properly (Verification) Be sure the water you are using is 22 to 25C Ensure the scale is free of drafts  Place a wt. on and verify it provides a consistent reading

 Set the Micropipetter to deliver 2 uL on a 2 to 20 uL pipetor or 25 uL on the 20 to 200 uL pipetor  Place a weigh boat on the calibrated balance  Add a few drops of water to the weigh boat  Tare the balance by pressing the zero button

 Pipet the appropriate volume and dispense it into the weigh boat.  Record your reading  Zero the balance (good practice to change tips each time)  Pipet the appropriate volume and dispense it into the weigh boat.  Record your reading  Repeat until you have 5 readings

 Set the Micropipetter to 20 uL for uL pipet or 200 for 20 to 200 uL pipet  Zero the balance  Pipet the appropriate volume and dispense it into the weigh boat.  Record your reading  Repeat until you have 5 readings  Add together the 5 volumes (n = 5) at each volume test point and divide the total by 5 to determine the mean volume, v. This value is expressed as either milliliters or microliters:

 Calculations: Calculate the mean weight of water  Density of water at 22C = g/mL  1 uL would weigh how many mgs? mgs Convert the mean water weight to the mean volume weight  Mean Volume of water = Mean weight / density

Calibration Procedure  Mean of 5 weights / mgs i.e. if the weights of 2 uL setting were 2.001, 2.001, 2.003, 2.002, mg What is the mean weight?  What is the mean volume of water?  2.018/ = What is the accuracy of the pipetor?  / x 100 = 100.3% or.3% error

Preventive maintenance of pipets  Wipe the pipet with alcohol if it gets contaminated with solution  Store properly which is in a stand if possible as you do not want the shaft part of the pipet to get bent from laying on a bench  Preventive Maintenance  Pipettes should receive preventive maintenance on an annual basis or whenever the instrument fails to perform as expected. Preventive maintenance involves cleaning the instrument thoroughly, inside and out, inspecting each part for wear and tear,

Designing the Test Plan  ISO requires that manufacturers test pipettes at the nominal (highest) volume, a mid point (typically 50%) and at a low point (usually 10%) of the pipette’s range. 10 replicate samples are aspirated and dispensed at each of the three volume test points. The mean of the 10 samples is used to determine accuracy, and the standard deviation of the 10 samples is used to determine the precision of the pipette. Users may choose to use fewer samples; a minimum of 3 are required. The Rainin Technical Report 9804 Comparison of Ten vs. Four Weighing Method discusses the statistical differences between the two methods in terms of the risk of rejecting a conforming, or passing, pipette when using the four sample method. The study found that when instruments are provided preventive maintenance prior to testing, the risk of a false reject is reduced to less than 1%. Of course performing preventive maintenance prior to testing performance eliminates the possibility of obtaining As Found performance data.  For the purposes of this training, we will use what is known as a 3x5 calibration structure: 3 test volumes with 5 replicate samples at each test volume. We will use test volumes of 10%, 50% and 100% of nominal volume.

Kalibrasyon  Analytical Balance with appropriate resolution (consult Table 1)  Weighing Vessel with a height to diameter ratio of at least 3:1.  Thermometer with accuracy of +/-.4 degree C.  Barometer or local barometric pressure  Single Channel Adjustable Volume Pipette  A supply of tips  DI or Double Distilled water  Beaker for holding DI or Double Distilled water

 Calibrating the Pipette  First ensure that all test equipment, the pipette, the tips and the DI or Double Distilled water have been allowed to equilibrate to room temperature. Care should be taken to ensure that the analytical balance is set up on a stable laboratory bench in an area free from direct sunlight, drafts and the vibrations of neighboring equipment.

 Place the weighing vessel in the center of the weigh pan of the analytical balance.  2. Use the thermometer to ascertain and record the temperature at the time of the test.  3. Use the Barometer to ascertain and record the barometric pressure at the time of test. While barometric pressure is required for the weight to volume conversion, its effect on volume is insignificant at 5 or 6 decimal places and therefore the barometric pressure reported for the local area at Weather.com is also sufficient.  4. Select and affix an appropriately sized tip to your pipette.  5. Dial the instrument down to the low volume (10% of nominal volume) test point.

 6. Taking care to immerse the tip 2-3 mm into the DI test water, hold the pipette in a near vertical position and aspirate and dispense 3 to 5 throw away samples to introduce humidity into the tip and shaft of the pipette.  7. Aspirate the first test sample and drag the tip along the side of the vessel to remove any excess sample on the outside of the tip.  8. Dispense the sample into the weighing vessel on the weigh pan.  9. Record the balance reading.

 10. Repeat steps 7 though 9 to obtain the remaining 4 samples at the low volume.  11. Discard the used tip, replace it with a fresh one, and dial up the volume to the mid point test volume (50% of nominal volume).  12. Repeat steps 6 though 9 until all 5 samples are obtained at the mid point test volume.  13. Discard the used tip, replace it with a fresh one, and dial up to the nominal volume test point.  14. Repeat steps 6 through 9 until all 5 samples are obtained at the nominal test point volume

Hesaplamalar  Convert each measurement in mass m by applying the Z correction factors from Table 3 at the mean temperature and barometric pressure measured and recorded at the time of the test using this equation:

Hesaplama  Add together the 5 volumes (n = 5) at each volume test point and divide the total by 5 to determine the mean volume, v. This value is expressed as either milliliters or microliters:

Hesaplamalar  Calculate the inaccuracy (systematic error) es for each test point using the following equation where vs is the target (selected test) volume:  To calculate the inaccuracy in percentage form, use this equation :

Hesaplamalar  Calculate the imprecision (random error) Sr for each test point using this equation:

Hesaplamalar  The random error can be expressed as a percentage, by the coefficient of variation, by using the following equation where vO is the nominal volume:

Hesaplamalar  Compare the results to your pre- determined tolerances to determine the performance of the pipette. Instruments that are not performing within your selected tolerances will require Preventive Maintenance and adjustment. Most manufacturers will sell adjustment tools and provide instructions for making the necessary adjustments to bring the instrument back within tolerance.

Pipetler(Pipettes)  Laboratuvarda ölçülen sıvının hacmini taşımak ve aktarmak için tasarlanmış malzemelerdir.  Laboratuvarlarda üç tip cam pipet kullanılır. Hacimsel Mohr Serolojik  Mekaniksel

Pipetler  Serolojik ve Mohr pipetleri farklı kullanıldıkları için farklı işaretlenirler.  Serolojik pipetler taşımak için (to delivered) tasarlanmıştır. Ölçülen hacmi hassas bir şekilde aktarabilmesi için son bir kez üflenirler.  Mohr pipetler TC yani içinde sıvı ihtiva edecek şekilde tasarlanmıştır.

İşaretlemeler  Aktarma pipetlerinin üst kısmına bir etiket veya renki bir halka vardır.  TC pipetlerin üst kısımlarında halka yoktur. Bazen hacimler renkli skala çizgileri ile belirlenmiş olabilir.

Hacimsel Pipetler  Belirli fix bir hacmi transfer etmek üzere tasarlanmışlardır..  Pipetlerin üzerinde belirli bir hacim için tek bir ölçü çizgisi vardır.  Örneğin 5 mL hacimsel pipetin üzerinde tek bir hacim çizgisi var ise bu pipet tek hacimli bir pipettir ve 5 mL den başka hacmi ölçmez ve kullanılamaz..

Hacimsel Pipetler  Tam olarak tek bir hacmi transfer etmek için dizayn edilmişlerdir. Hacim pipetin üst kısmında gösterilir.  Pipetin üst kısmında cama kazınmış bir halka bulunur.  Sıvı bu halkaya kadar çekilir ve pipetleme menisküsün alt çizgisi tam bu halka üzerinde iken serbest bırakılır.  İkinci bir kaba bu hacmi transfer etmek için pipetin ucu kabın iç cidarına temas ettirilir ve sıvının pipetten serbest akışı beklenir.

Serolojik Pipetler- TD  En genel kullanımı olan pipetlerdir ve pipet üzerinde TD yani aktarım için tasarlandığına dair işaret bulunur.  Belirli sıvı hacmini transfer etmek için ayarlanmışlardır. has been calibrated "to deliver" a specified volume of liquid.  These pipets have no base mark, the graduations continue onto the tip and are graduated to deliver which means that ALL the measured liquid in the pipet must be delivered.  0.1 mm aralıklı 5 mL lik bir serolojik pipet 0.1 mL den 5 mL ye kadar 0.1 mm çözünürlüklü olan herhangi bir hacmi ölçme için kullanılmaktadır. 0.2 mL den daha küçük hacimleri ölçmek otomatik pipet ile ölçülebildiği halde.

Serolojik Pipetler- TD  TD or “üflemeli” pipetler pipet ucuna kadar derecelendirilmiştir ve pipetin üst kısmında kazınmış veya reklendirilmiş bir halka bulunmaktadır. Graduated to deliver, there is no base mark.  In this type of pipet the marking near the tip of the pipet should be the same as the total volume listed near the top of the pipet

Serological - TD  Pipets with double rings are designed to be "blown out" by pushing a small amount of air out of the pipet, completely emptying it.

Mohr – TC – to contain  A pipet with this marking has been calibrated to contain a specified volume of liquid.  These pipets have a single painted or frosted ring at the top and are allowed to simply drain with the tip placed against the side of the receiving vessel.  To accurately transfer fluid with this type of pipet, the meniscus must be precisely on a calibration mark both at the beginning and at the end of a transfer.

Mohr - TC  To accurately transfer fluid with this type of pipet, the meniscus must be precisely on a calibration mark both at the beginning and at the end of a transfer.  Near the top of this type of pipet you will find the total volume indicated and the size of the smallest gradations (i.e., 5 mL in 1/10, the total volume of the pipet is 5 mL, and it is graduated in 1/10 mL increments).

Proper Use  When filling a pipet, the tapered end is held beneath the surface of the liquid at all times.  The liquid is drawn into the pipet by suction until the level is equal to or greater than the volume of liquid to be delivered.  Since serological pipets are labeled with the zero mark at the top of the pipet you will need to subtract the amount you are going to pipet from the total volume of the pipet to determine the exact mark to fill the pipet to.  For example if you are going to pipet 6.5 mL using a 10 mL pipet you would fill the pipet to the 3.5 mL mark. To determine the total volume of the pipet look near the top of this type of pipet.

What volume does this pipet contain?

Proper Use  When reading the volume, ALWAYS view the pipette dead-on at eye level with the pipette held vertically, perpendicular to the ground.  Pipettes are designed to be used with a hand pump or bulb, of which there are many varieties.  Never use your mouth with a pipet!

Mechanical or Automatic  Mechanical pipets can be set to draw and dispense different volumes or be preset to deliver an exact volume.  Used to accurately deliver very small volumes, microliters, of liquid.  Although 0.1 mL could be delivered by a serological pipet most labs use mechanical, or automatic pipets.  A volume of 0.1 mL is equal to 100 microliters

Mechanical or Automatic  Mechanical pipets are operated by depressing the plunger.  On the downward stroke of the plunger there are TWO stops.  The first offers firm resistance, and the second is a hard stop.  To take up a volume in the pipet, place a tip on the end of the pipet.  Depress the plunger to the first stop and insert into the sample to be transferred.  Draw the liquid into the pipet by SLOWLY releasing the plunger.  To dispense the liquid from the pipet, place the tip of the pipet into the opening of the well and slowly depress the plunger all the way to the second stop.  When the liquid has been dispensed withdraw the pipet tip from the well BEFORE releasing the plunger.

Glassware  Glassware can be divided into 2 groups Non-volumetric glassware  Beaker  Flask Volumetric Glassware  Volumetric Flask  Graduated Cylinder

Beaker  Used for transferring liquid to another container or to transfer a small amount of reagent for use in procedures.  Volume is not accurate, just an estimate.  NEVER PLACE A REAGENT IN ANOTHER CONTAINER WITHOUT LABELING THE CONTAINER FIRST.

Erlenmeyer Flask  Features a conical base, a cylindrical neck and a flat bottom.  They are marked on the side (graduated) to indicate the approximate volume of their contents.  This is NOT used for ACCURATE measurement

Erlenmeyer Flask – an FYI  Like many other common pieces of glassware, Erlenmeyer flasks could potentially be used in the production of illegal narcotics.  In an effort to restrict such production, some U.S. states (including Texas) have begun requiring permits to purchase such glassware, including Erlenmeyer flasks, as well as chemicals identified as common starting materials.

Graduated Cylinder  For rapid measurement of liquid volume.  They are generally more accurate and precise for this purpose than flasks.  This is a semi-accurate liquid measuring vessels.

Reading the Volume  10 mL has approx 6.62 mL  100 mL 52.7 mL  25 mL has 11.5 mL

Volumetric Flask  A volumetric flask is used to measure very precisely one specific volume of liquid (100 ml, 250 ml, etc., depending on which flask you use).  This flask is used to prepare a solution of known concentration.  To make up a solution, first dissolve the solid material completely, in less fluid than required to fill the flask to the mark.  After the solid is completely dissolved, very carefully fill the flask to the mL mark.  The top is then sealed and the flask is inverted several times to mix.

References  Pipets and Pipetting:  Use of Serological Pipets  BIO 121 Introduction  Glassware  Lab Equipment  Volumetric Flask  Measuring Volume Using a Graduated Cylinder a learning activity

Test Room Condition  Temperature : Maintained at 20 ± 1 °C and during calibration ± 0. 5 °C stability  Humidity Maintained 35% to 85%  Prefer to maintain at 60 ± 10%  Stabilization Volumetric instruments and test water  1 to 2 hours

Temizleme Cleaning of volumetric glassware  Internal surface of volumetric vessels shall be cleaned sufficiently before the calibration  Oil or grease is removed by suitable solvent  The vessel should be nearly filled with an aqueous solution of a soap less detergent and shaken vigorously. It should then be repeatedly rinsed with distilled water  Be sure that vessel is cleaned before commence calibration

 Prior to any calibration test make sure that all instruments reached the ambient temperature in the laboratory  During the calibration maximum temperature variation in the laboratory should not exceed ±0.5 o C.  Set or read a meniscus against a reference line or scale  Be sure that the vessel or weighing bottle and the distilled water are at room temperature.

Kalibrasyon Prosedürü2  Fill distilled water up to the required volume  Weigh the testing container with distilled water.  Measure the temperature of distilled water using the thermometer.  Repeat the above step three times and record in the data recording form.  Repeat the above steps for cylinders at least at four different volumes

Calibration Procedure –Flask & Cylinder (contain) 1. Weigh the empty testing container and record the value 2. Fill distilled water up to the required volume 3. Weigh the testing container with distilled water. 4. Measure the temperature of distilled water using the thermometer. 5. Repeat the above step 2 through 4 three times and record in the data recording form. 6. Repeat the above step 2 through 5 for cylinders at least at three different volumes

Calibration Procedure –Pippet (Delivered) 1. Weigh a empty container and record the value 2. Fill pipette with distilled water up to the index mark 3. Any water on the outside of the tip is removed with filter paper after the filling is completed 4. Transfer the distilled water to empty vessel, weighted in step (1) 5. Weigh the vessel with distilled water. 6. Measure temperature of distilled water using the thermometer 7. Repeat the above steps three times and record in the data recording form.

Calibration Procedure – Burette (deliver) 1. Weigh empty container and record the value 2. Fill burettes with distilled water up to the index mark 3. Any water on the outside of the tip is removed with filter paper after the filling is completed 4. Transfer the distilled water to empty vessel, weighed in step (1) 5. Weigh the vessel with distilled water 6. Measure temperature of distilled water using the thermometer. 7. Repeat the above steps three times and record in the data recording form. 8. Repeat the above steps 1 through 7 for Burettes at least three different levels

 Principle of calibration - volumetric glass ware  Z values are given in Tables B6, B7 and B8 in ISO 4787 : 2010 for different types of glass at common air pressure Vs temperature Bouncy effect Temperature effect

Notasyon  R L- is the balance reading of vessel with water reference liquid in grams  R E - is the balance reading of empty vessel in grams  Ρ a - is the density of air, in grams per milliliter  ρ b - when weighing in air as though the density of the weight was 8.0 g/ml.  ρ w - is the density of water at t o C, in grams per milliliter.  ϒ - is the coefficient of cubical thermal expansion of the material of which the item of glassware, tested is made in reciprocal degrees Celsius,  t - is the temperature of the water used in testing in degrees Celsius.

Termal Genleşme Katsayıları Material Coefficient of cubical thermal expansion ϒ -1 °C Borosilicate glass Borosilicate glass Soda-lime glass 27

Density of water – Table B4 ISO 4787  Temperature Density (g/ml)  °C ρ w   

Data Recording  Use spread sheet for data recording  Record Temperature, Humidity, Barometric pressure  Record final data in a data recording sheet  Review from a third party  Prepare the final calibration certificate