Lab 4:
Preparation of Standard Curves
Standard curve
Standard curves are used to determine the concentration of substances. They are
obtained by relating a measured quantity to the concentration of the substance of
interest in "known" samples, i.e. Standards of known concentration. These
standards provide a reference to determine unknown concentrations. Thus amounts
chosen of standards need to span the range of concentrations expected to be found
in the "unknown" sample concentration.
The quantity (assay measurements as i.e. luminescence, radioactivity, fluorescence,
and optical density of various known concentrations of a substance) graphed on yaxis and standard concentrations on x-axis. Data analyzed by fitting a line on curve
(see Figure below).
To determine the unknown concentration of a substance in a sample (with same
assay as for standards used), intersect across the assay measurement on y with
standard concentration, and down to x. The concentration of substance in unknown
sample is the value on x.
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Task
Graphing standard curves and obtaining accurate patient and control values from
them.
Objectives
Upon completion of this exercise, the student will be able to:
1. Properly set up a standard curve whether using linear or semi-log paper.
a. Absorbance vs. concentration
b. % transmittance vs. concentration
2. Using the prepared curve, determine the concentrations for control and patient
specimens.
Materials
1. Linear graph paper
2. log graph paper
3. Absorbance and % transmittance values for standards, controls and patients.
Principle
Many laboratory tests require the outcome of a carefully controlled chemical
reaction be evaluated or read in a photometer (colorimeter or spectrophotometer).
Since these instruments are capable of only measuring the amount of light being
allowed to pass through the cuvette, their readout devices display % of light
transmitted or mathematically derived absorbance. One method of obtaining
concentration from % transmittance or absorbance is through the use of a standard
curve.
For our purposes, standard curves are defined as a graphs with absorption or %T
plotted on the Y axis, and increasing concentrations of standard along the X axis. If
Beer’s Law is followed, the resulting line representing absorbance vs concentration
will be straight.
A standard curve is constructed after obtaining the %T/Abs readings from a number
of solutions of known concentration (standards) used in a reaction or procedure.
After the readings are obtained each is plotted on semi-log (% transmittance) or
linear (absorbance) paper against the corresponding concentration. If the procedure
follows Beer's Law, the points plotted will generally lie such that a straight line can
be drawn through them. The concentration of controls and other unknowns (patient
samples) can be determined by locating their %T/Abs reading on the line, then
dropping an imaginary line down from that point to intersect the concentration axis.
Once a standard curve is developed for a particular test method on a particular
spectrophotometer, it should be checked periodically to determine that it is still
good. A new curve should be constructed when there is a change in: ! reagent lot
numbers
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Methodology/ procedure
an instrument parameter (change bulb, optics cleaned, etc.)
Once the curve is drawn, a number of things must be considered to determine its
acceptability.
The majority of the curve’s points should be on or close to the line. There could be
many reasons for a point not being on the line. If the standards are formed from a
series of dilutions, the accuracy of the dilutions must be suspect. Calculations of the
dilutions and spectrophotometer errores are other possibilities. Whether or not the
curve passes through the point of origin (the “0"), varies with the procedure. If
Beer’s law is followed and the procedure is linear at the lower concentrations, the
curve’s line generally goes through the zero.
Procedure I
Basic Standard Curve Characteristics
1. Neatness counts. Preparing a good standard curve takes time and practice. A
sharp pencil should be used during the early construction period.
2. Use the X axis for concentration. Determining how to space between the
individual concentrations is done by trial and error and will also depend on the
individual procedure.
3. The Y axis is labeled either %T (semi-log paper) or Absorbance (linear paper). The
amount of spacing for absorbance readings is often times determined through trial
and error.
4. Centered on top of the graph (such that it doesn't interfere with the readings)
should be the name of the analyte measured (i.e., glucose).
5. On the upper right portion of the graph (again, such that it doesn't interfere)
should be labeled as follows:
 Your Name , Date
 Analyte/Procedure
 Instrument
 Wavelength
6. The following is an abbreviated list of errors or problems encountered by students
in the past. It is provided for you to consider as pitfalls to avoid. You should keep
this/these page(s) handy when preparing your chemistry lab curves.
a. Bottom of Y axis did not start at 0.000.
b. Compressed Y or X axises.
c. Uneven spacing of Y or X axises.
d. Not labeling correctly/in the right place.
e. Drawing curve point to point.
f. Required information is missing/in the wrong place.
g. “Fat” pencil lines/double/smeared lines.
h. Making dots on curve’s line for unknown’s absorbance value.
I. Drawing dotted lines on graph representing how the concentration of unknown
was determined.
j. Drawing circles around dots on the curve line.
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Procedure II
Preparing a Hemoglobin Concentration Standard Curve
• Hemoglobin (Hb) is the standard abbreviation for hemoglobin, the oxygencarrying pigment and predominant protein in the red blood cells. Hemoglobin is
the protein that carries oxygen from the lungs to the tissues and carries carbon
dioxide from the tissues back to the lungs
• hemoglobin molecule consists of four polypeptide chains: two alpha chains and
two beta chains,The α and β globin chains are very similar in structure and each
one of them is liked with a heme molecule.
Measurement of hemoglobin
The Cyanmethemoglobin Method for Hb determination is the reference method.
Principle:
•
Whole blood is diluted in a solution of potassium Ferricyanide and
potassium cyanide.
•
The Hb is oxidize to methemoglobin by the potassium Ferricyanide.
•
The potassium cyanide then converts the methemoglobin to
cyanmethemoglobin.
•
The absorbance of the cyanmethemoglobin at 540 nm is directly
proportional to the Hb concentration.
•
Sulfhemoglobin is not converted to cyanmethemoglobin; therefore, it
can not be measured by this method.
Hb (Fe++) K3Fe (CN)6
methemoglobin (Fe+++ )
KCN
Cyanmethemoglobin
Procedure of standard curve
1. Create a standard curve, using a commercially available cyanmethemoglobin
standard which, has constant concentration 25g/dl, the following dilutions
should be made to get the line between the concentration & the absorbance of
the standard using also drab
2. kin reagent as shown:
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Hb
concentration
g/dl
Absorbance
reading
Volume of St
/ ml
V of Drabkin
reagent / ml
0
0
0
5
5
0.125
1
4
10
0.250
2
3
15
0.375
3
2
20
0.500
4
1
2. Transfer the dilutions to cuvettes. Starting with the blank, measure the
absorbance on a spectrophotometer at 540 nm.
3. Plot absorbance on the y-axis and the Hb concentration on the x-axis. The Hb
concentrations of the patients’ samples and controls can be read from this
standard curve.
Standard Curve
0.5
0.4
0.3
0.2
0.1
•
0
5
10
15
Concentration
5
20
Calculation
Slope = abs (st)
Con (st)
1
Slope
= Con (st) = Factor
abs (st)
C (x)
= Con (st) X abs (x)
abs (st)
C (x)
=
Factor
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X abs (x)