7.

CALIBRATIONS,
STANDARDIZATIONS AND BLANK
CORRECTIONS
JSS/Oct.19.2022
7. Calibrations, Standardizations and Blank
Corrections
• The relationship between the measured signal, Smeas, and the
absolute amount of analyte is
Smeas = knA + Sreag 5.1
or the relative amount of analyte in a sample
Smeas = kCA + Sreag 5.2
where nA is the moles of analyte, CA is the analyte’s concentration, k is
the method’s sensitivity, and Sreag is the contribution to Smeas from
constant errors introduced by the reagents used in the analysis.
Calibrating Signals
• An object’s true weight in vacuo, Wv, is related to its weight in air, Wa,
by the equation

• where Do is the object’s density, Dw is the density of the calibration

weight, and 0.0012 is the density of air under normal laboratory
conditions (all densities are in units of g/cm3).
EXAMPLE 5.1
A 10-mL volumetric pipet was calibrated following the procedure just outlined, using a balance calibrated
with brass weights having a density of 8.40 g/cm3. At 25 °C the pipet was found to dispense 9.9736 g of
water. What is the actual volume dispensed by the pipet?
SOLUTION
At 25 °C the density of water is 0.99705 g/cm3. The
water’s true weight, therefore, is
and the actual volume of water dispensed by the
pipet is
If the buoyancy correction is ignored, the pipet’s
volume is reported as
introducing a negative determinate error of –0.11%. [(10.003-10.014)/10.014] x 100 = -0.11%
Reagents Used as Standards
• Primary Reagent
• A reagent of known purity that can be
used to make a solution of known
concentration.
• Example: K2Cr2O7
• Secondary Reagent
• A reagent whose purity must be
established relative to a primary
reagent.
• Example: NaOH
• Reagent Grade
• Reagents conforming to standards set
by the American Chemical Society.
Single-Point versus Multiple-Point
Standardizations
• Single-point standardization
• Any standardization using a single
standard containing a known
amount of analyte.
• A single standard containing a
known concentration of analyte,
CS, is prepared and its signal,
Sstand, is measured.
• The value of k is calculated as
Single-Point versus Multiple-Point
Standardizations
• Multiple-point standardization
• Any standardization using two or more standards containing known amounts
of analyte.
External Standards
• A standard solution containing a known amount of analyte, prepared
separately from samples containing the analyte.
• A quantitative determination using a single external standard was
described at the beginning of this section, with k given by equation
5.3.

• Once standardized, the concentration of analyte, CA, is given as

EXAMPLE 5.2
A spectrophotometric method for the quantitative determination of Pb2+ levels in blood yields an Sstand of
0.474 for a standard whose concentration of lead is 1.75 ppb. How many parts per billion of Pb2+ occur in a
sample of blood if Ssamp is 0.361?
Solution
Equation 5.3 allows us to calculate the value of k for
this method using the data for the standard

Once k is known, the concentration of Pb2+ in the

sample of blood can be calculated using equation 5.4
 Normal calibration curve
• A multiple-point external standardization is accomplished by
constructing a calibration curve, two examples of which are
shown in Figure 5.3.
• Since this is the most frequently employed method of
standardization, the resulting relationship often is called a
normal calibration curve.
• When the calibration curve is a linear (Figure 5.3a), the slope of
the line gives the value of k.
• In Figure 5.3b, the value of k is greatest when the analyte’s
concentration is small and decreases continuously as the
amount of analyte is increased.
• The value of k at any point along the calibration curve is given by
the slope at that point.
• In either case, the calibration curve provides a means for relating
Ssamp to the analyte’s concentration.
EXAMPLE 5.3
A second spectrophotometric method for the quantitative determination of Pb2+ levels in blood gives a
linear normal calibration curve for which

What is the Pb2+ level (in ppb) in a sample of blood if Ssamp is 0.397?

Solution
To determine the concentration of Pb2+ in the sample
of blood, we replace Sstand in the calibration equation
with Ssamp and solve for CA
Matrix matching
• A proportional determinate error is •
introduced when differences between
the two matrices cannot be ignored.
• This is shown in Figure 5.4, where the
relationship between the signal and
the amount of analyte is shown for
both the sample’s matrix and the
standard’s matrix.
• Adjusting the matrix of an external
standard so that it is the same as the
matrix of the samples to be analyzed.
• This is known as matrix matching.
Standard Additions
• A standardization in which
aliquots of a standard solution
are added to the sample.
• This is known as the method of
standard additions.
• The simplest version of a
standard addition is shown in
Figure 5.5.
*Aliquot is a portion of a solution.
Standard Additions
• The following two equations relate Ssamp and Sspike to the concentration of
analyte, CA, in the original sample

• where the ratios Vo/Vf and Vs/Vf account for the dilution.
• As long as Vs is small relative to Vo, the effect of adding the standard to the
sample’s matrix is insignificant, and the matrices of the sample and the spiked
sample may be considered identical.
• Under these conditions the value of k is the same in equations 5.5 and 5.6.
Solving both equations for k and equating gives
EXAMPLE 5.4
A third spectrophotometric method for the quantitative determination of the concentration of Pb2+ in blood
yields an Ssamp of 0.193 for a 1.00-mL sample of blood that has been diluted to 5.00 mL. A second 1.00-mL
sample is spiked with 1.00 µL of a 1560-ppb Pb2+ standard and diluted to 5.00 mL, yielding an Sspike of 0.419.
Determine the concentration of Pb2+ in the original sample of blood.
SOLUTION
The concentration of Pb2+ in the original sample of
blood can be determined by making appropriate
substitutions into equation 5.7 and solving for CA.
Note that all volumes must be in the same units, thus
Vs is converted from 1.00 µL to 1.00 × 10–3 mL.
Standard additions
• It also is possible to make a standard
addition directly to the sample after
measuring Ssamp (Figure 5.6).
• In this case, the final volume after the
standard addition is Vo + Vs and
equations 5.5–5.7 become
EXAMPLE 5.5
A fourth spectrophotometric method for the quantitative determination of the concentration of Pb2+ in
blood yields an Ssamp of 0.712 for a 5.00-mL sample of blood. After spiking the blood sample with 5.00 µL of
a 1560-ppb Pb2+ standard, an Sspike of 1.546 is measured. Determine the concentration of Pb2+ in the
original sample of blood.
Solution
The concentration of Pb2+ in the original sample of
blood can be determined by making appropriate
substitutions into equation 5.9 and solving for CA.
EXAMPLE 5.6
Starting with equation 5.6, show that the equations for the slope, y-intercept, and x-intercept in Figure 5.7(a)
are correct.
Equation 5.6

Figure 5.7
Slope = kCs/Vf

y-intercept = kCaVo/Vf

x-intercept = -CAVo/Cs
EXAMPLE 5.6…
Solution
We begin by rewriting equation 5.6 as

which is in the form of the linear equation Y = y-intercept + slope × X

where Y is Sspike and X is Vs.
The slope of the line, therefore, is → Slope = kCs/Vf
and the y-intercept is → y-intercept = kCAVo/Vf
The x-intercept is the value of X when Y is 0, or
EXAMPLE 5.7
A fifth spectrophotometric method for the quantitative determination of the concentration of Pb2+ in blood
uses a multiple-point standard addition based on equation 5.6. The original blood sample has a volume of
1.00 mL, and the standard used for spiking the sample has a concentration of 1560 ppb Pb2+. All samples
were diluted to 5.00 mL before measuring the signal. A calibration curve of Sspike versus Vs is described by
Sspike = 0.266 + 312 mL–1 × Vs
Determine the concentration of Pb2+ in the original sample of blood.
Solution
Equation 5.6
To find the x-intercept we let Sspike equal 0 0 = 0.266 + 312 mL–1 × (x-intercept)
and solve for the x-intercept’s absolute value, x-intercept = /-0.266 / 312 ml-1/
= 8.526 x 10-4 mL
Thus, x-intercept = 8.526 x 10-4 mL
= CAVo/CS
= CA (1.00 mL) / 1560 ppb)
= CA (6.410 x 10-4 mL/ppb)
CA = (8.526 x 10-4 mL) / (6.410 x 10-4 mL/ppb)
CA = 1.33 ppb
Internal standard
• A standard, whose identity is different from the analyte’s that is
added to all samples and standards containing the analyte.
If a solution contains an analyte of concentration SA = kACA
CA, and an internal standard of concentration, CIS, SIS = kISCIS
then the signals due to the analyte, SA, and the
internal standard, SIS, are
where kA and kIS are the sensitivities for the
analyte and internal standard, respectively.
Taking the ratio of the two signals gives SA/SIS = kA/kIS x CA/CIS
SA/SIS = K x CA/CIS eqn. 5.10
SA/SIS = K x CA/CIS
K = (CIS/CA) (SA/SIS)stand eqn. 5.11
CA = (CIS/K) (SA/SIS)samp
EXAMPLE 5.8
A sixth spectrophotometric method for the quantitative determination of Pb2+ levels in blood uses Cu2+ as
an internal standard. A standard containing 1.75 ppb Pb2+ and 2.25 ppb Cu2+ yields a ratio of SA/SIS of 2.37.
A sample of blood is spiked with the same concentration of Cu2+, giving a signal ratio of 1.80. Determine the
concentration of Pb2+ in the sample of blood.
Solution
Equation 5.11 allows us to calculate the value of K K = (CIS/CA) (SA/SIS)stand eqn. 5.11
using the data for the standard

The concentration of Pb2+, therefore, is

EXAMPLE 5.9
A seventh spectrophotometric method for the quantitative determination of Pb2+ levels in blood gives a
linear internal standards calibration curve for which

What is the concentration (in ppb) of Pb2+ in a sample of blood if (SA/SIS)samp is 2.80?
Solution
To determine the concentration of Pb2+ in the sample
of blood, we replace (SA/SIS)stand in the calibration
equation with (SA/SIS)samp and solve for CA
Linear Regression and Calibration Curves
Linear Regression and Calibration Curves
Blank Corrections
• In discussing ways to standardize a
method, we assumed that an
appropriate reagent blank had
been used to correct Smeas for
signals originating from sources
other than the analyte.
• In one study, 12 analytical chemists
were asked to evaluate a data set
consisting of a normal calibration
curve, three samples of different
size but drawn from the same
source, and an analyte-free sample
(Table 5.3).
Blank Corrections
At least four different approaches for correcting the signals were used by the
participants:
(1) Ignore the correction entirely, which clearly is incorrect;
(2) use the y-intercept of the calibration curve as a calibration blank, CB;
(3) use the analyte-free sample as a reagent blank, RB; and
(4) use both the calibration and reagent blanks.

Equations for calculating the concentration of analyte using each approach

are shown in Table 5.4, along with the resulting concentration for the analyte
in each of the three samples
Blank Corrections
Blank Corrections
• Unfortunately, neither the calibration blank nor the reagent blank can correct for
bias due to analyte–matrix interactions because the analyte is missing in the
reagent blank, and the sample’s matrix is missing from the calibration blank.
• The true method blank must include both the matrix and the analyte and,
consequently, can only be determined using the sample itself.
• One approach is to measure the signal for samples of different size and
determine the regression line from a plot of signal versus the amount of sample.
• The resulting y-intercept gives the signal for the condition of no sample and is
known as the total Youden blank. The total Youden blank is a blank that corrects
the signal for analyte–matrix interactions.
• This is the true blank correction.
• The regression line for the sample data in Table 5.3 is
• Ssamp = 0.009844 × Wx + 0.185
giving a true blank correction of 0.185.
• Using this value to correct the signals gives identical values for the concentration
of analyte in all three samples (see Table 5.4, bottom row).
 7. CALIBRATIONS,
STANDARDIZATIONS AND BLANK
CORRECTIONS
JSS/Oct.19.2022
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