Advice on Units and Standard Curve
PDF File for Printing: Help on Units
UNITS OF AMOUNTS AND CONCENTRATION:
Amounts are always given in moles, mmoles, µmoles, nmoles, etc. (symbol can be shortened
to "mol" -- for example µmol). You can not write 10-3 µmol because this is
nmol. You can always write 10-3 mol, but there is no need to since mmol is simpler and a
better way to represent your data. Please refrain from using exponentials for amounts.
When the molecular weight of a substance is unknown (i.e. for a mixture of proteins), the
amount can be given as the mass -- g, mg, µg, ng, etc.
Concentration is always given in M, mM, µM, nM, etc. (symbol "M" means moles
per liter and so "µM" means micromoles per liter). You can not write 10-3 µM
because this is nM. You can always write 10-3 M, but there is no need to since mM is
simpler and a better way to represent your data. Please refrain from using exponentials
for concentrations. For substances of unknown molecular weight (i.e. for a mixture of
proteins), the concentration can be given as g/liter, mg/ml, µg/ml, ng/µl, etc.
If you need to calculate an amount from a volume and concentration:
vol x conc = amount.
For example, (60 µM) x (1.0 ml) = 60 nmol or 0.06 µmol.
An easy way to think about this is: 60 µM = 60 nmol/ml so 1 ml of it contains 60 nmol.
If you need to derive a concentration when you mix a fixed volume of a given concentration
with another volume, use the final volume you arrive at to calculate the final
concentration:
Final Concentration = (Initial Concentration X Initial Volume)/Final Volume
C2 = (C1 x V1)/V2
Standard Curves:
Standard Curves are used for quantitative analysis of unknown substances by comparison
to a "standard".
For example, in evaluating the protein concentration of a sample, the amount in a given
volume of unknown protein solution can be determined by comparison to the amounts of a
"standard protein" using a dye that reacts with both the standard protein and
the unknown. So determining the concentration of protein in the unknown solution is a two
step process: 1) find out how much protein is present in fixed volumes of the unknown
using the standard curve prepared with known amounts of the standard protein; and 2)
calculate the concentration of protein in the unknown by dividing the protein amount by
the volume of unknown used.
This concept is illustrated below:
Calculations for the Unknown Protein in this example:
| Volume of Unknown (ml) | Absorbance | Amount of Protein (mg) from Standard Curve | Concentration (mg/ml) |
| 0.01 | 0.011 | Too Low to be Useful | ---- |
| 0.1 | 0.210 | 2.8 | 28 |
| 0.2 | 0.360 | 4.8 | 24 |
| 0.3 | 0.650 | 8.6 | 29 |
| 0.5 | 0.910 | Too High (Off Curve) | ---- |
- The Volume of the Unknown (ml) is "given" information from the protocol
instructions in the text.
Absorbance is what you determined in the experiment in the lab. - Amount of Protein (mg) is determined from the standard curve as shown by the lines drawn for specific volumes of unknown in the example standard curve shown above.
- Concentration is calculated by dividing the Amount of Protein (mg) by the Volume of Sample used to obtain that amount of unknown protein (ml) and the units of concentration are mg/ml.
- Once all the valid concentrations are calculated, then the average is taken. In this
example,
average = (28 + 24 + 29)/3 = 27 mg/ml (Remember to use the appropriate number of significant figures)
NOTE:
You can also calculate the Amount of Protein (mg) in the samples using the Equation of the Line for the Standard Curve:
Equation of Line = Linear Regression (Go To: Explanation of Linear Regression)
Amount of Unknown Protein in mg = (Absorbance - y intercept)/slope in absorbance/mg
protein
(note this is the inversion of the normal result obtained for the graph where y = a + bx,
when y = absorbance, a = y intercept, and b = slope of line in terms of absorbance/amount
of protein in mg).
For this example, equation of line = Amount of Unknown Protein (mg) = (specific
Absorbance - 0.000)/0.075
For sample volume = 0.1 ml, Absorbance = 0.21; therefore
Amount of Unknown Protein (mg) = (0.210 - 0.000)/0.075 = 2.8 mg
Now using the volume of this sample, the concentration = 2.8 mg/0.1 ml = 28 mg/ml
Note: Not all standard curves go through the origin, which means that the Y-intercept may be a significant value in making your calculation of the amount of protein.
Go To: Lecture 1 or Part 2 of Lecture 1