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Analytical Standards and Calibration Curves

Group 3
Andi Tenri Nurwahidah
Nining Sintia Dewi
Dewi Puspita
Hamdan Hidayat

Analytical Standards and Calibration

There are many analytical methods

that require some form of calibration
before use in quantitative analysis.
Although not the only way, a very
important and popular method for
this calibration is by means of a
6.1 Simple Linear
The general equation for a linear function, expressed in
concentration terms is given by Eq. 6.1a.

Y mC b

There, Y = meausred parameter

C = concertration of analyte
m = proportionally constant
b = a constant term (often considered as the
experimental blank value)

A blank value can be defined as a measurement made on a

sample, following an estabilished procedur, but where the
analyte was not added to the sample.
Fig.6.1a shows Eq.6.1a in graphical terms

Fig.6.1a graphical representation of simple linear

The proportionalty constant m is goven by the slope
of the graph illustrated in Fig. 6.1a and ca be
calculated as below.
m tan MN LM

When measuring KM and MN, always remember to

relate these the scale on youre axes, and do not
measure purely in terms of distance.

Fig. 6.1b shows a tyical calibration

mC b plot for an
analytical method obeying the equation
Calculate the slope of the line.

Fig. 6.1b Calibration plot for an analytical method

From Fig. 6.1b as labelled

m = MN/LM = 20/0.20 = 10 x 102

Determinate errors are defined as measureable error which in theory have a

definite value and which can be allowed for during the analysis. They may be
caused by the analyst, the instrument, or the method.
In a colorimetric determination of formaldehyde (methanal) the following
result were used to construct a calibration graph :

Mass of formaldehyde Absorbance

0 0.036
20.0 0.154
40.0 0.273
60.0 0.389
80.0 0.507
100.0 0.626

Is The methode exhibiting a determinate error?

In order to see if there a determinate error the blank value must be subtraced
from all the sample values and then a graph of corrected absorbance against
mass of formaldehyde should be constructed.

Mass of formaldehyde Corrected absorbance

20.0 0.118
40.0 0.237
60.0 0.353
80.0 0.471
100.0 0.590
Fig.6.1c identification of determinate error
As all of the points lie on straight line which can be extrapolated through the
origin, the method has been shown, within wxperimental error, to have no
determonate error. In fact equation now becomes that originally shown in Eq.

mC a

It should be obvious that a positive intercept may arise from the presence of
the analyte in the reagents used in the analytical method. It may be less
obvious how a negative intercept can be occur. One way, is in
spectrophotometric measurment on solutions, when unmatchedd cells are
used the reference cell absorbs more rediation than the sample cell.
SAQ 6.1a

Fig. 6.1d Linear calibration plot for use in SAQ6.1a

Indeterminate error is errors that arise from uncertainties in a
measurement, they are unknown and cannot be cntrolled by the
analyst. The effect will produce a scatter of result for replicate
measurements which can be assesed only by statistical test.

Measurement statistics and computation for a more

comprehensive description and discussion
6.1.1. Linear Regression Analysis
The simplest procedure for linear regeression analysis is
that known as the method of least squares.
It use a mathematical formula to calculate the values of
m and b in our equation, given that we have a set of
data relating Y to C. The two values are calculated by
using Eqs. 6.1c and 6.1d below :
C Y n CY
m ...........(6.1c)
( C ) 2 n C 2
2 Y

b ............(6.1d )
( C 2 ) n C 2

Where : n = number of result available

= the sum of the designated functions of all of its n
When applying the method of least squaresyou will
make a number assumptions

That a linear relationship does indeed exist between the

measured parameter Y and the analyte concentration C.
That no significant error exist in the composition of the
standard or in C. Thus the deviation of the points from
an exact straight line are entirely due to the
indeterminate error in the value of Y.
That there are no pieces of data, which fall outside the
normal statistical pattern, included in the calibration.
These pieces of bad data, as they arre sometimes
called, should be rejected, either after visual
examination or after the use of statistical tests (see
ACOL Unit : Measurement, Statistics and Computation).
The accuracy of a least-squares fit is a function of the
number of data-points used in the calibration. A m
inimum of six points is recommended. However in this
unit, to reduce the tedium of the calibration, fewer
result than the recommended number have been used.
The set of data listed in the table below relates to an
analytical method obeying the following linear
Y mC b Y C
0.33 4.0
0.50 8.0
For example :
0.75 12.0
093 16.0
Compare the two straight lines
Looking at Eq. 6.1c and 6.1d we see that the values
we need to calculate initially are :
C 2 , CY
and then :
0.33 4 16 1.32
0.50 8 64 4.00
0.75 12 144 9.00
0.93 16 256 14.88
Y = 2.51 C = 40 C = 480 CY = 29.2
( C 2 ) 1600
(40 x 2.51) (4 x 29.2) (40 x 29.2) (480 x 2.51)
m b
1600 (4 x 480) 1600 (4 x 480)
100.4 116 .8 1168 1204.8

1600 1920 1600 1920
16.4 36.8

320 320
In order to construct the best straight line by using
these values we return to the equation for that
line, ie Y mC b
We select the maximum value of C used in the
calibration (ie C = 16), and calculate the value of Y
which corresponds to this value
We now have wo point through which we can
construct our straight line.
Y = 0.115, C = 0
Y = 0.93, C = 16

Fig. 6.1e. Best straight line through data points

6.2 Non-Linear Calibration
Though most comparative obey a linear
relationship as ready described iin section 6.1. this
obedience to linearity is often own only over a
relatively small concentration range. Go outside
range and negative deviation may accur as
ilustrated in fig.

Fig. 6.2a. Negative deviation

From zero concentration to concentration (X) the linearity iis
obeyed, but above concentration (X) the system is
showing negative deviation.
Curvature does not affect the validity of a calibration plot,
but does mean that more care must be taken in
preparation, by using more standards to predict the
curve accurately.
The effect of curvature in calibration plots is often met in
spectrophotometric methods involving emission or
absorption of radiation.
The curvature arises from instrumental features of from
chemical changes that occur t higher concentration and
will be fully explained in a later unit.
The main problem with analytical methods which exhibit
curved calibration plots is that method are not open to
abriged calibration, standard addition, or internal
standard procedure.
6.2.1 Transformation of Non-linear
into Linear Function
In analytical chemistry there are a number of
useful techniques where the function relating Y to
C is not initially linear,
eg :
Y k10 ............(6.2a )
10 aY kC............(6.2b)
Where : Y =measured parameter
C = concentration
a and k are contants
Eq. 6.2a tell us that there is a reduction in the
parameter Y with increase in concenration, with
the opposite being true for Eq. 6.2b. The equation
are shown graphically in Fig. 6.2b.
Fig. 6.2b. Graphically representation of Eq. 6.2a and
We can however, Create a linear relationship
from both of these equations by taking the logs of
both sides. Y k10 aC
Thus , taking logs for gives
log Y log k aC............(6.2c)
aY log k log C

10 aY kC
and taking logs for gives
aY log k log C............(6.2d )

The plots relating to Eq. 6.2c and 6.2d are

ilustrated in Fig. 6.2c below.
Fig 6.2c. Graphically representation of Eq. 6.2c and
The availability of modern scientific calculators has
simplified considerably calibration involving
logarithmic function and their subsequent
The table below gives a set of calibration data for an
analytical method obeying the relationship.
Y k m log C
Concentration of Measured parameter
analyte, C (ppm) Y
100 0/150
200 0.173
300 0.186
400 0.195
If a sample solution gave a value of Y of 0.179, what
was the concentration of the analyte in the sample ?
Page 123
Are told that the relationship is that Y is
proportional to log must first calculate log C
C log C Y
100 2.00 0.150
200 2.30 0.173
300 2.48 0.186
400 2.60 0.195
We can now plot a Y vs log C graph
From our graph we can
now find the log C
value corresponding to
a Y value of 0.179. This
is log C = 2.38(5)

By taking the antilog of

this we can obtain C

Antilog of 2.38(5) = C
= 243

Fig. 6.2d.Determinate of analyte concertation

SAQ 6.2a
An analytical method is known to obey the
Y be mC
Does this method exhibit a relationship where by either
(i) Y increases with increasing concentration
(ii) Y decreases with increasing concentration

tempt to answer part (A) of this SAQ before continuing to par

The following set of data was obtained from an analytical me

thought to obey the experimentalrelationship quoted above
SAQ 6.2a

2,26 2,00

1,97 4,00

1,67 6,00

1,45 8,00

1,23 10,00
SAQ 6.2a
What are you conclusions regarding this hypothesis ?

s the hypothesis
i)Definitely true
ii)Probably true
iii)Definitely false
iv)Probably false ?