Accuracy vs. Precision
- These help us understand our measement
- Accuracy: How close a measurement or attempt is to the actual or target value
- Precision: How consistent our result are regardless of proximity to the actual or
target
- Comparison:
+ Accuracy is measuring near true value
+ Precision is getting consistent results
- It’s important that measuring devices are accurate and precise, if not result can be
misleading and fatal
Direct vs. Indirect Meserments
- Both types of measurements are routinely used in science
- Direct measuments: this is measuring the thing you are actually measuring
+ Example: FInding the thickness of a leaf with calipers
+ Counting the number of cells in a culture with a haemocytometer
- Indirect measurment: this is measuring something by measuring something else.
+ Example: Determing the number of cells in a solution by counting the number
of colonies in a dibute overnight culture
+ Determing the concentration of a solution by measuring the
absorbance of its metabolite
Pipetting
- Gilson Micropipette usage:
+ Use with plastic tip to transfer 0.2 microliters to 1000 microliters or 1 mililiter
+ Different pipette have a label on top indicates the maximum volume transfer
- P2: 0.2 - 2 microliters
- P20: 2- 20
- P200: 20- 200
- P1000: 200-1000
+ Setting volume:
- P2: (for ex: 152 means 1.52 micrliters)
- P20: ( for ex: 152 means 15.2 )
- P200: ( 152 means 152)
- P1000 ( 052 means 520) the 100= 1000 microliters which is the
maximum of the setting volume
- Setting volume:
+ Pipette tip:
- P20 and P200 use the same yellow tips
- P1000 use the larger white tips
+ Pipette volume:
- P20 set at 2 microliters require less push than the P20 set at 20
+ Common tip to avoid:
- Never use the pipette without the tip as the liquid should never enter the body
of pipette
, - Do not use the pipette that pass the volume maximum
- if the tip is falling down use the new one
- Eppendorf pipetting: has 3 stops
Principles of Spectrophotometry:
Absorbance
- Absorption Spectroscopy: using light to probe matter. Look for how a sample
absorbs light of a particular wavelength which related to concentration
+ In experiment, we look for a wavelength that is absorbed by a specific
sample. The light has an incoming power (P in). High power light or high
intensity light will have a lot of photons coming in. We also have a solution
with high concentration that can absorb the light, therefore, the P out is small.
+ A lot of photons get absorbed through the sample-> absorption spectroscopy
+ The ratio bwt the input power and output power related to sample
concentration
- Percent transmittance (%T): percentage of light that gets transmitted through our
sample = P out/ P in
- The spectrometry that can measure the output and input light intensity.
- Absorbance (A): linear related to the concentration. Does not have a unit. A= 2-
log(%T)= log(1/T)
- The transmittance and absorbance is inverslly related. 100% absorbance we will get
0% transmittance.
- Do different experiments by absorbance spectrum to know which suitable
wavelength. We scan through a lot of wavelengths. If a solution is blue, the red is
absorbed. FInd the lambda max.
Principles of Spectrophotometry: How
does a Spectronic 20 work?
- Energy source-> monochromator (prism to separate the different colour of light) to
select the wavelength -> get absorbed through the sample -> hit the detector.
- Turn the knob in the nanometer for the visible light to control the wavelength of light
the sample is exposed to
- The phototube in the detector is coated with photoemisive that can measure the light
intensity
- How to operate Spec 20 to generate an ansorption spectra of a dye called DCPIP:
+ A basic spectrophotometer with analog dials and manual setting of 0%
transmittance and blanking
+ The Specs 20s accept glass test tubes in the sample chamber (requires a
high volume of sample) about 3mL volume. Can ửok for one sample at a time
- These help us understand our measement
- Accuracy: How close a measurement or attempt is to the actual or target value
- Precision: How consistent our result are regardless of proximity to the actual or
target
- Comparison:
+ Accuracy is measuring near true value
+ Precision is getting consistent results
- It’s important that measuring devices are accurate and precise, if not result can be
misleading and fatal
Direct vs. Indirect Meserments
- Both types of measurements are routinely used in science
- Direct measuments: this is measuring the thing you are actually measuring
+ Example: FInding the thickness of a leaf with calipers
+ Counting the number of cells in a culture with a haemocytometer
- Indirect measurment: this is measuring something by measuring something else.
+ Example: Determing the number of cells in a solution by counting the number
of colonies in a dibute overnight culture
+ Determing the concentration of a solution by measuring the
absorbance of its metabolite
Pipetting
- Gilson Micropipette usage:
+ Use with plastic tip to transfer 0.2 microliters to 1000 microliters or 1 mililiter
+ Different pipette have a label on top indicates the maximum volume transfer
- P2: 0.2 - 2 microliters
- P20: 2- 20
- P200: 20- 200
- P1000: 200-1000
+ Setting volume:
- P2: (for ex: 152 means 1.52 micrliters)
- P20: ( for ex: 152 means 15.2 )
- P200: ( 152 means 152)
- P1000 ( 052 means 520) the 100= 1000 microliters which is the
maximum of the setting volume
- Setting volume:
+ Pipette tip:
- P20 and P200 use the same yellow tips
- P1000 use the larger white tips
+ Pipette volume:
- P20 set at 2 microliters require less push than the P20 set at 20
+ Common tip to avoid:
- Never use the pipette without the tip as the liquid should never enter the body
of pipette
, - Do not use the pipette that pass the volume maximum
- if the tip is falling down use the new one
- Eppendorf pipetting: has 3 stops
Principles of Spectrophotometry:
Absorbance
- Absorption Spectroscopy: using light to probe matter. Look for how a sample
absorbs light of a particular wavelength which related to concentration
+ In experiment, we look for a wavelength that is absorbed by a specific
sample. The light has an incoming power (P in). High power light or high
intensity light will have a lot of photons coming in. We also have a solution
with high concentration that can absorb the light, therefore, the P out is small.
+ A lot of photons get absorbed through the sample-> absorption spectroscopy
+ The ratio bwt the input power and output power related to sample
concentration
- Percent transmittance (%T): percentage of light that gets transmitted through our
sample = P out/ P in
- The spectrometry that can measure the output and input light intensity.
- Absorbance (A): linear related to the concentration. Does not have a unit. A= 2-
log(%T)= log(1/T)
- The transmittance and absorbance is inverslly related. 100% absorbance we will get
0% transmittance.
- Do different experiments by absorbance spectrum to know which suitable
wavelength. We scan through a lot of wavelengths. If a solution is blue, the red is
absorbed. FInd the lambda max.
Principles of Spectrophotometry: How
does a Spectronic 20 work?
- Energy source-> monochromator (prism to separate the different colour of light) to
select the wavelength -> get absorbed through the sample -> hit the detector.
- Turn the knob in the nanometer for the visible light to control the wavelength of light
the sample is exposed to
- The phototube in the detector is coated with photoemisive that can measure the light
intensity
- How to operate Spec 20 to generate an ansorption spectra of a dye called DCPIP:
+ A basic spectrophotometer with analog dials and manual setting of 0%
transmittance and blanking
+ The Specs 20s accept glass test tubes in the sample chamber (requires a
high volume of sample) about 3mL volume. Can ửok for one sample at a time
