BIO LAB PART 3 ANALYTICAL TECHNIQUES

P1000 micropipette measures from

100 (or 200 depending on the model) - 1000 uL

EQUATION FOR ABSORBANCE

2-log(10)(% Transmittance)

ACCURACY

A description of how close a measurement is to the true value of the quantity measured.

Micropipettes measure both ------ and ------ in the lab,

ACCURACY AND PRECISION

WHITE LIGHT consists of light with wavelengths from :

About 400 nm (violet) to about 700 nm (red)

The linear relationship between the concentration of a colored solution and its light absorbance is known as the ______.

Beer-Lambert Law

What micropipette(s) could be used to measure out 175μL of liquid?

P200 AND P1000

THE LINEAR RELATIONSHIP BETWEEN THE GLUCOSE CONCENTRATION AND THE INTENSITY OF THE COLOR PRODUCED BY THE CHEMICAL REACTION. - THIS IS KNOWN AS THE:

STANDARD CURVE

What is the name of the machine we will use in today's lab to measure the amount of light that is absorbed by a solution?

Spectrophotometer

TRANSMITTENCE

The spectrophotometer allows individual wavelengths of light to pass through the sample and the wavelengths of light that are not absorbed go through the sample and arrive at a photoelectric tube that determines HOW MUCH LIGHT GOT THROUGH. For ex: if no light gets through, there will be 0% transmittance - If all light got through the transmittance would be 100%

HOW TO FIND AN IDEAL WAVELENGTH VIA MAXIMUM ABSORBANCE STEP 5:

Then, if we plot the concentration of glucose (the independent variable) on the x-axis and the absorbance of the colored solution (y-axis) of a graph, we get the standard curve. Then we can use the standard curve to graphically determine the concentration of glucose in a sample from a patient.

ABSORBANCE

This is what we are most interested in, as it tells us HOW MUCH light was absorbed or taken in by the sample

How to determine if someone is diabetic?

- If their readings for fasting plasma glucose levels are 126 mg/dL or higher - Two "random" glucose readings of 2200 mg/dL or higher.

HOW TO FIND AN IDEAL WAVELENGTH VIA MAXIMUM ABSORBANCE STEP 2:

- There should be an inverse relationship between the color of a solution and the wavelength of light it absorbs.... FOR EX: A blue solution maximally absorbs light with wavelengths at the red end of the visible spectrum (700 nm) whilst a red solution maximally absorbs light with wavelengths at the blue end of the spectrum (400 nm). YOU CAN EASILY ESTIMATE THE RANGE OF WAVELENGTHS A SOLUTION ABSORBS BEST BY REFERRING TO A COLOR WHEEL

1 uL (microliter) is how many 10^-6 L

0.000001 L

100μL is equivalent to:

0.1mL

1,000,000 uL (microliters) is how many Liters

1 L

1,000 uL (microliters) is how many millimeters

1 mL

P20 micropipette measures from

1-20 uL

1 uL (microliter) is how many 10^-3 mL

10^3 mL (0.001mL)

What volume of liquid would a P200 micropipette at this setting (pictured below-picture says 155) measure out?

155 μL

P200 micropipette measures from

20-200 uL

HOW TO FIND AN IDEAL WAVELENGTH VIA MAXIMUM ABSORBANCE STEP 3:

Before we can measure the concentration of glucose in a plasma sample, we have to KNOW THE LINEAR RELATIONSHIP BETWEEN THE GLUCOSE CONCENTRATION AND THE INTENSITY OF THE COLOR PRODUCED BY THE CHEMICAL REACTION.

Which image below depicts high accuracy but low precision?

C.

What color-producing chemical will be used in today's lab to determine the concentration of glucose solutions through absorbance readings?

Chromogen

HOW TO FIND AN IDEAL WAVELENGTH VIA MAXIMUM ABSORBANCE STEP 1:

Determine which wavelength of light a colored solution will maximally absorb. -We want to use this wavelength in our subsequent work to get the most accurate and precise results. - This is accomplished by placing a colored solution in the spectrophotometer, and then passing light through the sample in wavelength increments of 25 nm.

If the results of a patient's two "random" glucose readings are 196 mg/dL and 200mg/dL, this person meets the criteria of diabetes mellitus. TRUE OR FALSE

FALSE

There is a(n) __________ relationship between the color of a solution and the wavelength of light it best absorbs.

INVERSE

HOW TO FIND AN IDEAL WAVELENGTH VIA MAXIMUM ABSORANCE STEP 6:

Lastly, as aforementioned, if we plot the curve with the independent variable (concentration) on the x-axis, and the dependent variable (absorbance) on the y-axis, and we determine the mathematical equation (y=mx+b) of the straight line of the standard curve, we can then mathematically determine the concentration of the patient sample from its absorbance (by solving for x). - The linear relationship between the concentration of a colored solution and its light absorbance is known as the BEER-LAMBERT LAW.

Blue-colored objects absorb which wavelength of light the most?

RED

HOW TO FIND AN IDEAL WAVELENGTH VIA MAXIMUM ABSORBANCE STEP 4:

To make a standard curve, the first step is to take a solution of known glucose concentration and dilute it, producing a series of solutions of decreasing glucose concentration. - Then the color producing chemicals (chromogen) are added, the color is generated, and then the spectrophotometer is used to determine the absorbance of each tube with a solution of known glucose concentration

Beer-Lambert Law

Used to relate the concentration of colored solutions to the amount of visible light they absorb.

PRECISION

a measure of how close a series of measurements are to one another

what is diabetes?

a metabolic disease in which the body's inability to produce any or enough insulin causes elevated levels of glucose in the blood.