2. Biochemistry Practical Dept.of Biochemistry Zhihong Li ( 李志红 ) PhD

3. REGULATIONS OF PRACTICAL Successful experiments usually depend on accurate managements. All students should comply with these regulations. 1.The students should come into the lab on time. It is prohibit being absent, late or leaving earlier. Anyone who is late for 15 minutes or more should be thought as absenteeism. 2. It is necessary to put on the work clothes before entering the lab. Not permitted to come into if the students are wearing vest, short pants or slipper. 3. Keep quiet and neat in the lab. Any actions, which are not concerned with your experiment, should be avoided.

4. 4. The students should know the content of practical, and write the objectives, principle and procedure on the report book in advance. (you can download the content from our website) 5. Carefully observe the experimental phenomena, record the experimental result and discuss the significance of the experiment. 6. Wash your hands in time after finished the experiment or touched the toxic chemicals. 7. Discard solid wastes in the provided waste containers, not in the sink! Rinse non-hazardous, water-soluble wastes such as small amounts of acids down the drain.

5. 8. Carefully and accurately operate the apparatus. It should be kept in good order after your finishing the operation. It should be compensated according to the cause that results in the damage of the apparatus. 9. Each student should clean the bench, take back the apparatus to Room 2509, treat with the trash, and leave off after the permission of the instructor. The students who are on duty should clean the lab after the experiment according to the duty arrangement sheet. 10. The monitor should collect the report books and hand over them to Room S-2514 (or S-2509) before next experiment.

6. Contents 1Volume transfer, accuracy and precision 2 Spectrophotometry & plotting of calibration curve 3 Serum total protein 4Enzyme inhibition test 5Alkaline phophatase 6Serum glucose test 7Final experimental operation exam

7. Final practical grade Final practical grade= –Daily performance (30%): report book, attendance, operation and so on. –Final operation exam (35%) –Final writing exam (35%) 1/3 absent, can not take part in the final exam.

8. Volume transfer, accuracy and precision BIO - 01

9. PURPOSE To learn how to handle pipettes, micropipetts and electronic analytical balance. To learn how to dispense solution properly. To learn how to dispose and analyze the experimental data correctly.

10. Part Ⅰ : PIPETTE Volumetric pipette has one calibration mark and is designed to deliver one fixed volume. Measuring Pipets include Serological and Mohr pipettes deliver various volumes to varying degrees of accuracy. The measuring pipette is calibrated along its length. Two types of pipette are commonly used in lab: Volumetric (or transfer) pipets Measuring pipets

11. TECHNIQUES FOR USING PIPETTES 1.Pipettes should be used only with Rubber Bulbs, mouth pipetting should not be allowed. 2.Squeeze the air out of the bulb and press the opening of the bulb against the opening of the pipet. Notice: the tip of the pipet must be kept under the surface of the liquid the entire time suction is applied or air will be sucked into the pipet.

12. 3.Fill the pipette above the calibration mark using a pipette bulb. 4.Quickly remove the bulb and place your index finger (not your thumb) over the end of the pipet.. 5.Adjust liquid level to the calibrating line during delivery. The lowest part of the meniscus, when it is sighted at eye level, should be level with calibration line on the pipette. Right eye level

13. 6. Tip the pipette against the beaker to remove any excess solution. 7. Reset the pipette tip against the wall of the container into which the solution is to be transferred and allow the solution to drain. Leave the pipette in this position for at least 10 seconds after all the solution appears to have drained out and touch the pipet tip to the side of the flask to remove any droplets. 8. Remove the pipet. DO NOT BLOW OUT THE SOLUTION REMAINING IN THE PIPET. The pipettes are calibrated in a manner that takes into account the solution which remains at the tip due to surface tension.

14. These pipettes are generally used in handling reagents and are considered not accurate enough to handle standard and sample.

15. Part Ⅱ : Micropipette Multi-channel pipette Single-channel pipette

16. FUNCTION OF THE MICROPIPETTE Micropipettes are used to transfer small liquid volumes.Micropipettes are used to transfer small liquid volumes. It is a precision instrument calibrated in microliters (μL). 1ml = 1000 (μL) It was invented in 1957 by Heinrich Schnitger in Germany.It was invented in 1957 by Heinrich Schnitger in Germany. These pipettes operate by piston-driven air displacement.These pipettes operate by piston-driven air displacement.

17. CAREFUL HANDLING OF THE MICROPIPETTE The micropipette must be handled with great care, to avoid damage. A damaged micropipette may produce imprecise measurements and this could affect the results of the experiment. Do not rough-handle or drop the micropipette.

18. Structure of micropipette Push botton handle tip ejector digital volume indicator tip ejector arm tip holder

19. Select proper Pipette: Micropipette have different volume range. P5000 1,000 ~ 5,000μl P1000 200 ~ 1,000 μl P200 50 ~ 200 μl P100 20 ~ 100 μl P20 2 ~ 20 μl P10 0.5 ~ 10 μl How to handle micropipette:

20. Micropipettes Micropipettes have 3 positions:Micropipettes have 3 positions: –Rest position. –First stop. –Second stop.

21. Volume Adjustment Knob: Digital Volume Indicator: Pipettors – 3 Volumes: Step 1: Set and read the Volume Operating the Micropipette Hold the micropipette in one hand and with the other hand, turn the volume adjustment knob (or shaft) until the volume indicator displays the desired volume. IF THE VOLUME ADJUSTMENT KNOB IS HARD TO TURN, STOP AND CALL THE INSTRUCTOR. DO NOT TRY TO FORCE THE KNOB TO TURN BECAUSE YOU WILL DAMAGE THE MICROPIPETTE. Do not adjust the micropipette volume above or below that recommended range (!!!).

22. Example of tip sizes: Attaching the disposable tip Operating the Micropipette-2 Step 2: Attach the Disposable Tip Select a box containing the correct size tips to be used. Place the pointed end of the micropipette shaft into one of the tips. Press down and twist slightly to ensure an airtight seal, then lift-up the micropipette with tip attached. YOU MUST NEVER USE THE MICROPIPETTE WITHOUT A TIP ATTACHED !!!

23. Operating the Micropipette-3 Step 3: Depress the Plunger to the First Stop Step 5: Draw up the sample Step 5: Pause To aspirate the sample into the tip, allow the pushbutton to return slowly and smoothly to the fully extended UP POSITION. NEVER LET THE PLUNGER SNAP UP! Wait a few seconds to ensure that the full volume of sample is drawn into the plastic tip. WAIT LONGER FOR LARGER VOLUMES or MORE VISCOUS ("SYRUP-LIKE") SUBSTANCES. Step 4: Immerse Tip in Sample

24. Operating the Micropipette-4 Step 6: Withdraw the Tip Remove the tip from the sample liquid. Be sure No liquid remain on the OUTSIDE of the tip. Visually examine the tip to make sure it contains reagent and that there are no bubbles present.

25. Operating the Micropipette-5 Step 8: Dispense the Sample Step 7: To dispense the solution from the pipette: a) Touch the tip end to the side wall of the receiving vessel. b) Depress the plunger to the FIRST STOP. c) Pause for at least one second. d) Press the plunger to the SECOND STOP (the second point, of greater resistance, at the bottom of the stroke) to expel any residual liquid in the tip (like "blowing out" a glass pipette). (a) Start Dispensing (b) 1st Stop = Dispense (c) 2nd Stop = Expel

26. Operating the Micropipette-6 Step 8: Withdraw the Pipette With the plunger fully depressed, withdraw the pipette from the receiving vessel carefully. Step 9: Release the Plunger Gently allow the plunger to return to the UP position. Step 11: Discard the Tip Step 10: Discard the tip by depressing the tip ejector button, as shown below. A fresh tip should be used for each sample to prevent sample carryover. Press ejector button to discard tip.

27. Step-wise Operation of the Micropipette Step-wise Operation of the Micropipette Set volume.Set volume. Attach disposable tip.Attach disposable tip. Depress the plunger to 1 st stop.Depress the plunger to 1 st stop. Immerse tip in sample and Draw up sample.Immerse tip in sample and Draw up sample. Withdraw the tip.Withdraw the tip. Dispense the sample by pushing the plunger to the 2 nd stop.Dispense the sample by pushing the plunger to the 2 nd stop. Withdraw the pipette and release the plunger.Withdraw the pipette and release the plunger. Discard the tip.Discard the tip.

28. Pippetting Guidelines and Precautions For optimal reproducibility, use the following pipetting procedures: (1) Consistent SPEED and SMOOTHNESS when you press and release the PLUNGER (2) Consistent pressure on the PLUNGER at the FIRST STOP (3) Consistent and sufficient IMMERSION DEPTH (4) Nearly VERTICAL POSITIONING of pipette (5) AVOID ALL AIR BUBBLES: Since the plastic pipette shaft can be damaged if liquids are drawn beyond the tip into the shaft. (6) NEVER lay the pipette on its SIDE nor INVERT the pipette if liquid is in the tip.

29. Part Ⅲ : Accuracy and Precision In Science, we want measurements to be both accurate and precise. What is the difference between them?

30. Accuracy and Precision Accuracy is a measure of rightness. –It means "capable of providing a correct reading or measurement." –It refers to how closely a measured value agrees with the actual value. Precision is a measure of exactness. –Precise means “repeatable, reliable, getting the same measurement each time.” –It refers to how closely individual measurements agree with each other.

31. Three targets with three arrows each to shoot. Can you hit the bull's-eye? Both accurate and precise Precise but not accurate Neither accurate nor precise How do they compare?

32. Error Sources Instrumental errors Natural errors Personal errors

33. Instrumental Errors Caused by imperfections in instrument construction or adjustment Examples – imperfect spacing of graduations, nominally perpendicular axes not at exactly 90°, level bubbles or crosshairs misadjusted … Fundamental principle – keep instrument in adjustment to the extent feasible

34. Natural Errors Errors caused by conditions in the environment that are not nominal Examples – temperature different from standard, atmospheric pressure variation, gravity variation, magnetic fields, wind and so on

35. Personal Errors Errors due to limitations in human senses or dexterity Examples – ability to read a micrometer, steadiness of the hand, estimate between graduations, … These factors may be influenced by conditions such as weather, insects, hazards, …

36. Some of the afore-mentioned errors (instrumental, natural, and personal) occur in a systematic manner and others behave with apparent randomness. They are therefore referred to as systematic and random errors.

37. Systematic Errors Sources: –Instrumental, physical and human limitations. Example: Device is out-of calibration. How to minimize them? –Careful calibration. –Best possible techniques. Are TYPICALLY present. Measurements are given as: Measurement + Systematic Error OR Measurement - Systematic Error

38. Random Errors ALWAYS present. Measurements are often shown as: Measurement ± Random Error Sources: –Operator errors –Changes in experimental conditions How to minimize them? –Take repeated measurements and calculate their average.

39. Absolute error : E = X i - X T Relative error : (1) Accuracy and error Where, Xi: measured value; X T : True value

40. (2) Precision and deviation standard deviation (SD or S) Relative Standard Deviation (RSD) or Coefficient of Variation (CV) Mean: X=

41. VolumeAccuracyPrecision ul error （ ul ） error （％） S.D. （ ul ） R.S.D. （ ul ） P20 2±0.1±5.0≤0.03≤1.5 5±0.1±2.0≤0.04≤0.80 10±0.1±1.0≤0.05≤0.50 20±0.2±1.0≤0.06≤0.30 P100 20±0.35±1.8≤0.10≤0.5 50±0.40±0.8≤0.12≤0.24 100±0.80±0.8≤0.15 P200 50±0.5±1.0≤0.20≤0.40 100±0.8 ≤0.25 200±1.6±0.8≤0.30≤0.15 P1000 200±3±1.5≤0.60≤1.30 500±4±0.8≤1.0≤0.20 1000±8±0.8≤1.5≤0.15 Accuracy and Precision of Micropipette

42. How to use electronic analytical balance Operation 1.Check to ensure that the horizontal position of the balance is level. Each balance is equipped with a level indicator. 2.Switch on /power 3.Tare,make scale “0.000” 4.Weight ： record the weight Attention Horizontal-level bubble gauge

43. Pipette 1) Prepare a electronic balance 2) Prepare 200 ml of distilled water 3) Prepare a glass beaker 4) Weigh the glass beaker and Tare out the balance (i.e., make the scale 0.000) 5 ） Select glass pipettes and rubber bulb 6 ） Dispense 3.8ml (or 1.6ml) distilled water in the beaker and read the balance and record the weight 7) Repeat 2~3 times and record the weight of each time 8 ） Calculate the Volume using the recorded weight and Temp-Density conversion table. 9 ） Calculate PRACTICE

44. 1) Prepare a electronic chemical balance 2) Prepare 200 ml of distilled water and measure the temperature 3) Prepare a glass beaker 4) Weigh the glass beaker and Tear out the balance (i.e., make the scale 0.000) 5) Dispense 550 µl (or 160 µl) distilled water in the beaker and read the balance and record the weight 6) Repeat 2~3 times and record the weight of each time. 7) Calculate the Volume using the recorded weight and Temp- Density conversion table. 8) Calculate Micropipette

45. Results Pipette Required volume (ml) 3.8 (or 1.6) Roll number number 1234 5678910101 1212 Measured Mass (g) Measured Volume (ml) Absolute Error Relative Error Mean of measured volume SD Micropipette Required volume (µl) 550 (or 160 ) Roll number number 12345678910101 1212 Measured Mass (g) Measured Volume (ml) Absolute Error Relative Error Mean of measured volume SD Volume (ml)= Mass (g) / Density (g/cm 3 ). Note the significant digits.

46. Table. the different density of water at different temperature T( 0 C)Density(g/cm 3 )T( 0 C)Density(g/cm 3 )T( 0 C)Density(g/cm 3 ) 00.999 868120.999 525240.997 326 10.999 927130.999 404250.997 074 20.999 968140.999 271260.996 813 30.999 992150.999 126270.996 542 41.000 000160.998 970280.996 262 50.999 992170.998 802290.995 973 60.999 968180.998 623300.995 676 70.999 929190.998 433310.995 369 80.999 876200.998 232320.995 054 90.999 869210.998 021330.994 731 100.999 728220.997 799340.994 399 110.999 632230.997 567350.994 059

47. Discussion How about the accuracy and precision of your operation (pipette and micropipette)? What are the precautions when you operate the pipette and micropipette?

48. Next experiment Spectrophotometry & Plotting of Calibration Curve