I.

About Graphs
What is a graph?
A graph is a diagram, as a curve, broken line, or series of bars, representing
various kinds of quantitative information and relationships, such as the successive
changes in a variable quantity or quantities.
Two-dimensional drawing showing a relationship (usually between two set
of numbers) by means of a line, curve, a series of bars, or other symbols. Typically,
an independent variable is represented on the horizontal line (X-axis) and
an dependent variable on the vertical line (Y-axis). The perpendicular axis intersect
at a point called origin, and are calibrated in the units of the quantities represented.
Though a graph usually has four quadrants representing the positive and
negative values of the variables, usually only the north-east quadrant is shown
when the negative values do not exist or are of no interest.
What are the types of graphs?
1. Line Graph
Line graphs are used when one variable (independent) affects another,
which is the dependent variable and it is useful for predicting in trends.
Graphical device that displays quantitative information or illustrates
relationships between two changing quantities (variables) with a line or curve
that connects a series of successive data points. A grouped line graph compares
a trend with one or more other trends, and shows if its rate of change is
increasing, decreasing, fluctuating, or remaining constant. Line graphs are the
most versatile and most extensively used family of graphs. Also called line
chart.

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2. Bar Graph
The data for this graph are non-numerical and discrete for at least one
variable. There are no dependent and independent variables. Axes may be
reversed to give a graph with the categories on the x axis.
A bar graph is a chart that uses bars to show comparisons between
categories of data. The bars can be either horizontal or vertical. Bar graphs with
vertical bars are sometimes called vertical bar graphs. A bar graph will have
two axes. One axis will describe the types of categories being compared, and
the other will have numerical values that represent the values of the data. It does
not matter which axis is which, but it will determine what bar graph is shown.
If the descriptions are on the horizontal axis, the bars will be oriented vertically,
and if the values are along the horizontal axis, the bars will be oriented
horizontally.
Bar graphs can therefore be drawn horizontally or vertically. This type of
graph is very useful for comparing two or more similar items.

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3. Pie Graph (Circle Graph)
Pie chart (or a circle chart) is a circular statistical graphic which is divided
into slices to illustrate numerical proportion. In a pie chart, the arc length of
each slice (and consequently its central angle and area), is proportional to the
quantity it represents. While it is named for its resemblance to a pie which has
been sliced, there are variations on the way it can be presented. The earliest
known pie chart is generally credited to William Playfair's Statistical
Breviary of 1801.

Pie charts are very widely used in the business world and the mass
media. However, they have been criticized,[4] and many experts recommend
avoiding them, pointing out that research has shown it is difficult to compare
different sections of a given pie chart, or to compare data across different pie
charts. Pie charts can be replaced in most cases by other plots such as the bar
chart, box plot or dot plots.

As with bar graphs, pie graphs are used when the data for one variable are
discrete (categories) and the data for the other variable are in the form of counts
(percentages or proportions).

A circle is divided according to the proportion of counts in each category.

Not suitable for data sets with a very large number of categories (more than 6).

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II. Line Graph

Importance of constructing and interpreting line graphs in analytical chemistry

The line graph helps to determine the relationship between two sets of values, with
one data set always being dependent on the other set.

Line graphs are drawn so that the independent data are on the horizontal a-axis (e.g.
time) and the dependent data are on the vertical y-axis. Line graphs are used to track
changes over short and long periods of time. There is some debate about the degree of
measurement between time points. Some say the data must be measured nearly
continually in order for the lines to be accurate representations. Others feel a monthly
measurement is sufficient, even though the line implies data at points where no
measurement was taken.

Line graphs are useful in that they show data variables and trends very clearly and
can help to make predictions about the results of data not yet recorded. They can also
be used to display several dependent variables against one independent variable. When
comparing data sets, line graphs are only useful if the axes follow the same scales.
Some experts recommend no more than 4 lines on a single graph; any more than that
and it becomes difficult to interpret.

Graph variables

Dependent Variables. A dependent variable is the variable being tested in a

scientific experiment. The dependent variable is 'dependent' on the independent
variable. As the experimenter changes the independent variable, the change in the
dependent variable is observed and recorded. When you take data in an experiment, the
dependent variable is the one being measured.
x and y are dummy variables, but often used by standard convention as dimensional
representations of the Cartesian plane. An independent variable is represented by the
abscissa (e.g. 'x'), and the dependendent variable as the ordinate (e.g. 'y').
As the value of the function y, where y = f(x) represents an instance where the value
of y is dependent upon the value of x, it would be correct to say that y is plotted against
x rather than the other way around.

4
Equation of a Line
Mathematical Equation
𝑦 = 𝑚𝑥 + 𝑏
Parameters
The slope is the number "m" that is multiplied on the x, and "b" is the y-
intercept (that is, the point where the line crosses the vertical y-axis). This
useful form of the line equation is sensibly named the "slope-intercept form".
Plotting data of a linear curve
a. Procedure
1. The first step in creating a graph using Microsoft Excel is entering the
data. The data should be in two adjacent columns with the x data in the
left column. In the figure below, I have labeled the columns but this is
not necessary to create the graph.
2. Position the cursor on the first X value (i.e., at the top of the column
containing the x values, or "Concentration” values), hold down the left
mouse button and drag the mouse cursor to the bottom Y value (i.e., at
the bottom of the column containing the y values, or "Absorbance"
values). All of the X-Y values should now be highlighted.
3. Click on the “Insert” tab at the top of the toolbar.
4. Under the “Chart” section, choose “Line”.
5. If you wish, you may label your graph (ie, put a title at the top of the
graph). You may wish to label the y-axis “absorbance” and the x-axis
“concentration”.
6. Next you want to determine the slope and intercept of your line. Move
the mouse cursor to any data point and press the left mouse button. All
of the data points should now be highlighted. Now, while the mouse
cursor is still on any one of the highlighted data points, press the right
mouse button, and click on “Add Trendline” from the menu that
appears.

5
 7. From within the "Trendline" window, select the type of “Trend /
Regression Type” you want – for a Beer’s Law plot the function should
be “Linear”.
8. At the bottom of the same menu, choose the option “Display Equation
on Chart”.
9. Click “OK”. A line and an equation should appear on the graph, as
shown below. Notice that this equation is in the format {y = mx + b},
and numerical values are provided for the slope and intercept. The value
of the intercept should be close to zero, a small number, but it may not
be exactly zero.
b. Line of best fit
A line of best fit is a straight line drawn through the center of a group of
data points plotted on a scatter plot. Scatter plots depict the results of gathering
data on two variables. The line of best fit shows whether these two variables
appear to be correlated and can be used to help identify trends occurring within
the dataset.
c. Methods of Least Squares (Linear Regression)
Using linear regression (LR) function of a scientific calculator
a. Procedure of encoding/key-in data
 You have to clear all data first.
Press"SHIFT" >>> Press "CLR">>>Press "3" (ALL) >>>
Display: Reset All >>> Press "=">>> Press "AC".
 Set the calculator to Linear Regression mode
Press "MODE" twice >>> Press "2" (REG) >>> Press "1" (Lin)
 Data Entry
Do data entry one by one with "x comma y" format
For example, you have data like this:
time (hours) Distance (m)
1 3
2 5
3 6.8

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 Then do data entry like this:
Press "1" >>> Press "," >>> Press "3" >>> Press "M+" >>> Display n = 1
Press "2" >>> Press "," >>> Press "5" >>> Press "M+" >>> Display n = 2
Press "3" >>> Press "," >>> Press "6" >>> Press ".">>> Press"8">>>Press
"M+" >>> Display n=3
 To know gradient, y-intercept, Correlation coefficient value
 Gradient (B)
Press "SHIFT" >>> Press "2" >>> Press ">" (next
button) twice >>> Press "2" (B) >>> Press "=" >>>
Display (for the above example: B= 1.9)
 y-intercept (A)
Press "SHIFT" >>> Press "2" >>> Press ">" (next
button) twice >>> Press "1" (A) >>> Press "=" >>>
Display (for the above example: B= 1.1333333)
 Correlation coefficient (r)
Press "SHIFT" >>> Press "2" >>> Press ">" (next
button) twice >>> Press "3" (r) >>> Press "=" >>>
Display (for the above example: r =0.9995386). If you
need R square, just use the square button.

Now, you get the equation : y = Bx+A >>> y = 1.9x +1.13333333 ( r = 0.9995386)
 Ah, you can check the entered data whether you entered the
correct data or no.
 Press "down" or "up" botton, you will see x1, y1, Freq1
to xn, yn, Freqn depending your data.
b. Determination of Linearity of data (Linearity check)
CALCULATE THE CORRELATION CO-EFFICIENT

[SHIFT] [1] [5: Reg] [3: r] [=]

r = .................................... r is very close to ......., hence there is a

.................................................................

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correlation between temperature and atmospheric pressure. Once it is determined
that r > 0,7 (positive or negative) we can calculate the linear regression line also
called the line of best fit, which will help us to predict future values. y = A + Bx
where A is the y-intercept and B is the gradient/slope

CALCULATE THE VALUE OF A

[SHIFT] [1] [5: Reg] [1: A] [=] A = ................

CALCULATE THE VALUE OF B

[SHIFT] [1] [5: Reg] [2: B] [=] B = ................ So the equation of the line of best fit
is y = ............... + ............... x

c. Calculating value of x for a given value of y

For calculating the value of x for a given value of y, first enter the
data for example;
X Y
10 1003
15 1005
20 1010
25 1011
30 1014
y=mx + b
y= 10.15x + 978.89
r= 0.9776
 Calculate the value of x when y is 1000
Input 1000 press SHIFT [2] find x the press [2] and press
“=” (x= 7.9915)
d. Calculate the value of y when x is 18
Input 18 press SHIFT [2] find ŷ then press [2] and press
“=” (y= 1008)

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III. Calibration curve
Definition
In analytical chemistry, a calibration curve, also known as a standard curve, is a
general method for determining the concentration of a substance in an unknown sample
by comparing the unknown to a set of standard samples of known concentration.

The calibration curve in chemistry is used primarily to correct the problems of

instrument calibration. The calibration curve in chemistry is used in conjunction with other
methods. Other popular methods include that by which the standard sample may be mixed
with the unknown, giving rise to an internal standard, which in turn may be described as a
chemical substance that is added in a constant amount to samples used for chemical
analysis.

The calibration curve in chemistry is a graph generated by experimental means. In

this, the concentration of solution is mapped on the x-axis, while the observable variable
is mapped on the y-axis.

Procedure

a) Prepare knowns amples of analyte covering convenient range of concentrations.

b) Measure the response of the analytical procedure.

c) Subtract average response of blank (no analyte).

d) Make graph of corrected response versus concentration.

e) Determine best straight line.

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Determination of value of unknown concentration of an analyte from a calibration curve

CALCULATING SOLUTION CONCENTRATION

Step 1. Construct a calibration plot of absorbance on the y-axis and concentration

on the x-axis for the standard solutions. The data points should fall along a reasonably
straight line. Two data points represent the absolute minimum, and more is better.

Step 2. Draw a “best-fit” straight line through the data points and extend the line to
intersect the y-axis. Choose two random points, not data points, on the line and determine
their x and y coordinates. Label these coordinates as (x1,y1) and (x2,y2).

Step 3. Calculate the slope, m, of the line according to the formula m = (y1 - y2) /
(x1 - x2). Determine the y-intercept, abbreviated b, by noting the the y-value where the line
crosses the y-axis. For example, for two random points on the line at coordinates (0.050,
0.105) and (0.525, 0.315), the slope is given by:

m = (0.105 - 0.315) / (0.050 - 0.525) = 0.440.

If the line crosses the y-axis at 0.08, then this value represents the y-intercept.

Step 4. Write the formula of the line of the calibration plot in the form y = mx + b.
Continuing the example from Step 3, the equation would be y = 0.440x + 0.080. This
represents the equation of the calibration curve.

Step 5. Substitute the absorbance of the solution of unknown concentration into the
equation determined as y and solve for x, where x represents concentration. If, for example,
an unknown solution exhibited an absorbance of 0.330, the equation would yield:

x = (y - 0.080) / 0.440 = (0.330 - 0.080) / 0.440 = 0.568 moles per liter.

10
Graph

Sample problems

Tooth enamel consists mainly of the mineral calcium hydroxyapatite,

Ca10(PO4)6(OH)2. Trace elements in teeth of archaeological specimens provide
anthropologists with clues about diet and disease of ancient people. Students at Hamline
University measured strontium in enamel from extracted wisdom teeth by atomic
absorption spectroscopy. Solutions with a constant total volume of 10.0 mL contained
0.750 mg of dissolved tooth enamel plus variable concentrations of added Sr. Find the
concentration of Sr.

Added Sr Signal
(ng/mL = ppb) (arbitrary units)

0 28.0

2.50 34.3

5.00 42.8

7.50 51.5

10.00 58.6

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IV. Standard Addition Method
Definition
The method of standard addition is a type of quantitative analysis approach often
used in analytical chemistry whereby the standard is added directly to the aliquots of
analyzed sample. This method is used in situations where sample matrix also
contributes to the analytical signal, a situation known as the matrix effect, thus making
it impossible to compare the analytical signal between sample and standard using the
traditional calibration curve approach.
Procedure
A typical procedure involves preparing several solutions containing the same
amount of unknown, but different amounts of standard. For example, five 25 mL
volumetric flasks are each filled with 10 mL of the unknown. Then the standard is
added in differing amounts, such as 0, 1, 2, 3, and 4 mL. The flasks are then diluted to
the mark and mixed well.
The idea of this procedure is that the total concentration of the analyte is the
combination of the unknown and the standard, and that the total concentration varies
linearly. If the signal response is linear in this concentration range, then a plot similar
to what is shown above is generated.

Standard Addition Curve

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 Preparation

Prepare a set of standard solutions to cover the expected range of analyte

concentrations. Fit a least squares regression line y = mx + b and calculate analyte
concentration in unknowns.

Interpretation of data
From a set of known samples is then used to make a standard curve, by plotting the
concentration on the x-axis, and assay measurement on the y-axis. If the data exhibits
a linear relationship the standard curve will be a straight line. The same assay is then
performed with samples of unknown concentration. To analyze the data, one locates
the measurement on the y-axis that corresponds to the assay measurement of the
unknown substance and follows a line to intersect the standard curve. The
corresponding value on the x-axis is the concentration of substance in the unknown
sample.

Illustration

Standard addition equation

Single addition method (Two-point data)

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Ssamp – signal of sample

Vo – initial volume of sample

Vstd – volume of standard

CA – concentration of the analyte

14
Multiple addition method (multiple data)

Sspike – signal of spike Vstd – volume of standard

KA – concentration constant Vf – final volume

CA – concentration of the analyte Cstd – concentration of standard

Sample problems

Application in spectroscopy

A third spectrophotometric method for the quantitative analysis of Pb2+ in blood

yields an Ssamp of 0.193 when a 1.00 mL sample of blood is diluted to 5.00 mL. A second
1.00 mL sample of blood is spiked with 1.00 mL of a 1560-ppb Pb2+ external standard and
diluted to 5.00 mL, yielding an Sspike of 0.419. What is the concentration of Pb2+ in the
original sample of blood?

Application in electrochemistry

The potential for an Fe3+/Fe2+ half-cell is +0.750 V relative to the standard

hydrogen electrode. What is its potential when using a saturated calomel electrode or a
saturated silver/silver chloride electrode?

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V. Spectrophotometric Studies of Complexes

Methods of continuous Variations

Description

A Job plot, otherwise known as the method of continuous variation or Job's method,
is a method used in analytical chemistry to determine the stoichiometry of a binding event.
The method is named after Paul Job and is also used in instrumental analysis and
advanced chemical equilibrium texts and research articles. Job first published his method
in 1928, while studying the associations of ions in solution. By plotting the UV
absorbance of a solution of Tl(NO3)/NH3 against the mole fraction of Tl(NO3), he
produced a graph which provided information about the equilibrium complexes present in
solution.

Sample problem

Six clean 50mL volumetric flasks were labeled A to F. The reagents were pipetted
into the flasks in the following order: iron solution, 5mL acetate buffer, 1mL
hydroxylamine, hydrochloride, 0.0007M phenathroline solution.

16
 The solutions in the volumetric flasks were diluted to mark and mixed thoroughly.
After 10 minutes, the absorbance of each solution was measured at 508nm using distilled
water as reference. Calculate the mole fraction of each component in the solution.

Mole-ratio Method

An alternative to the method of continuous variations for determining the

stoichiometry of metal-ligand complexes is the mole-ratio method in which the amount
of one reactant, usually the moles of metal, is held constant, while the amount of the other
reactant is varied.

17
Sample problem

Six clean 50mL volumetric flasks were labeled A to F. The reagents were pipetted
into the flasks in the following order: 2mL of iron standard solution, 5mL acetate buffer,
1mL hydroxylamine hydrochloride. The solution was mixed thoroughly. Then, 0.0007M
phenathroline solution was added. The solutions in the volumetric flasks were diluted to
mark and mixed thoroughly. After 10 minutes, the absorbance of each solution was
measured at 508nm using distilled water as reference. Calculate the number of moles
phenathroline and the number of moles of iron. What is the mole ratio? What is the
stoichiometry of the complex?

Slope-ratio Method

This method, used mainly in studying weak complexes, requires that the formation
reaction can be forced to completion with a large excess of either metal or ligand. Two sets
of solutions are prepared: The first contains various amounts of metal ion each with the
same large excess of ligand, while the second consists of various amounts of ligand each
with the same large excess of metal. For the reaction

xM + yL = MxLy

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Sample problem

Five clean 50mL volumetric flasks were labeled A to E. Into each flask, the reagents
were pipetted in the following order: Six clean 50mL volumetric flasks were labeled A to
F. The reagents were pipetted into the flasks in the following order: iron solution, 5mL
acetate buffer, 1mL hydroxylamine, hydrochloride, 0.0007M phenathroline solution. . The
solutions in the volumetric flasks were diluted to mark and mixed thoroughly. After 10
minutes, the absorbance of each solution was measured at 508nm using distilled water as
reference. Plot the concentration against absorbance. Plot concentration of phenathroline
against absorbance and determine their slopes. What is the stoichiometry of the complex?

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VI. Spectrophotometric Titrations
Description
The process of determining the quantity of a sample by adding measured
increments of a titrant until the end-point, at which essentially all of the sample has
reacted, is reached. The titration is followed by measuring the absorbance of radiation
in the range ultraviolet to near-infrared (0.1--2.5 \\mum) by the sample.

In chemistry, spectrophotometry is the quantitative measurement of the reflection

or transmission properties of a material as a function of wavelength. It is more specific than
the general term electromagnetic spectroscopy in that spectrophotometry deals
with visible light,near-ultraviolet, and near-infrared, but does not cover time resolved
spectroscopic techniques.

Spectrophotometry uses photometers, known as spectrophotometers, that can

measure a light beam's intensity as a function of its color (wavelength). Important features
of spectrophotometers are spectral bandwidth (the range of colors it can transmit through
the test sample), the percentage of sample-transmission, the logarithmic range of sample-
absorption, and sometimes a percentage of reflectance measurement.

A spectrophotometer is commonly used for the measurement of transmittance or

reflectance of solutions, transparent or opaque solids, such as polished glass, or gases.
Although many biochemicals are colored, as in, they absorb visible light and therefore can
be measured by colorimetric procedures, even colorless biochemicals can often be
converted to colored compounds suitable for chromogenic color-forming reactions to yield
compounds suitable for colorimetric analysis. However, they can also be designed to
measure the diffusivity on any of the listed light ranges that usually cover around 200 nm
- 2500 nm using different controls and calibrations. Within these ranges of light,
calibrations are needed on the machine using standards that vary in type depending on
the wavelength of the photometric determination.

20
Procedure

The end point for a titration of Cu2+ with EDTA can be identify, in the presence of
NH3 by monitoring the titrant’s absorbance at a wavelength of 745 nm, where the
Cu(NH3)42+ complex absorbs strongly. At the beginning of the titration the absorbance
is at a maximum. As we add EDTA the concentration of Cu(NH3)42+, and thus the
absorbance, decreases as EDTA displaces NH3. After the equivalence point the
absorbance remains essentially unchanged.

Graph

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Sample problem

Sketch the spectrophotometric titration curve for the titration of a mixture of 5.00 × 10–
3
M Bi3+ and 5.00 × 10–3 M Cu2+ with 0.0100 M EDTA. Assume that only the Cu2+–EDTA
complex absorbs at the selected wavelength.

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VII. Potentiometric Titrations
Description

Potentiometric titration is a technique similar to direct titration of a redox

reaction. It is a useful means of characterizing an acid. No indicator is used; instead the
potential is measured across the analyte, typically an electrolyte solution. To do this,
two electrodes are used, an indicator electrode (the glass electrode and metal ion
indicator electrode) and a reference electrode. Reference electrodes generally used are
hydrogen electrodes, calomel electrodes, and silver chloride electrodes. The indicator
electrode forms an electrochemical half-cell with the interested ions in the test solution.
The reference electrode forms the other half cell.

The overall electric potential is calculated as Ecell = Eind - Eref + Esol. Esol is the
potential drop over the test solution between the two electrodes. Ecell is recorded at
intervals as the titrant is added. A graph of potential against volume added can be
drawn and the end point of the reaction is halfway between the jump in voltage.
Ecell depends on the concentration of the interested ions with which the indicator
electrode is in contact. For example, the electrode reaction may be

Mn++ne−----->M

Procedure

Location of the end-point. It is obtained by plotting the successive values of the cell
emf on ordinate and corresponding values of volume of titrant added on the abscissa.
This gives an S-shaped curve. The central portion of this curve which shows the steeply
rising portion corresponds to the volume for the end point of the titration. When there
is a small potential change at the end point like in the titration of weak acid with strong
base, titration of very dilute solution etc, it is difficult to locate end point by this
method.

23
 Graph

Sample problem

Assume two solutions, one containing 0.01 M potassium dichromate, 1.0 x 10-6 M
chromium III from chromium III sulfate and 0.7 M hydrogen ion and one containing 0.012
M iron II sulfate and 1.0 x 10-6 M iron III from iron III sulfate. Answer each question
below assuming a temperature of 25 °C and that: A. The two solutions are contained in
separate beakers joined by a salt bridge with an inert electrode (such as platinum) immersed
in each solution. B. The two solutions are contained (no dilution) in a single beaker with a
saturated calomel electrode and an inert electrode (e.g., Pt) immersed in the solution.

Relevant information:

Cr2O7 2-/Cr3+: E° = +1.33 V

Fe3+/Fe2+: E° = +0.77 V

For SCE: Eref = +0.244 V

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VIII. Molecular weight determination using HPLC
Description of liquid chromatographic methods employed in molecular weight
determination
Partition chromatography was one of the first kinds of chromatography that
chemists developed.The partition coefficient principle has been applied in paper
chromatography, thin layer chromatography, gas phase and liquid–liquid
separation applications. The 1952 Nobel Prize in chemistry was earned by Archer John
Porter Martin and Richard Laurence Millington Synge for their development of the
technique, which was used for their separation of amino acids. Partition
chromatography uses a retained solvent, on the surface or within the grains or fibers of
an "inert" solid supporting matrix as with paper chromatography; or takes advantage of
some coulombic and/or hydrogen donor interaction with the stationary phase. Analyte
molecules partition between a liquid stationary phase and the eluent. Just as
in Hydrophilic Interaction Chromatography (HILIC; a sub-technique within HPLC),
this method separates analytes based on differences in their polarity. HILIC most often
uses a bonded polar stationary phase and a mobile phase made primarily
of acetonitrile with water as the strong component. Partition HPLC has been used
historically on unbonded silica or alumina supports. Each works effectively for
separating analytes by relative polar differences. HILIC bonded phases have the
advantage of separating acidic, basic and neutral solutes in a single chromatographic
run.
Normal–phase chromatography was one of the first kinds of HPLC that chemists
developed. Also known as normal-phase HPLC (NP-HPLC) this method separates
analytes based on their affinity for a polar stationary surface such as silica, hence it is
based on analyte ability to engage in polar interactions (such as hydrogen-
bonding or dipole-dipole type of interactions) with the sorbent surface. NP-HPLC uses
a non-polar, non-aqueous mobile phase (e.g. Chloroform), and works effectively for
separating analytes readily soluble in non-polar solvents. The analyte associates with
and is retained by the polar stationary phase. Adsorption strengths increase with
increased analyte polarity. The interaction strength depends not only on the functional
groups present in the structure of the analyte molecule, but also on steric factors. The

25
effect of steric hindrance on interaction strength allows this method to resolve
(separate) structural isomers.
The basic principle of displacement chromatography is: A molecule with a high
affinity for the chromatography matrix (the displacer) will compete effectively for
binding sites, and thus displace all molecules with lesser affinities. There are distinct
differences between displacement and elution chromatography. In elution mode,
substances typically emerge from a column in narrow, Gaussian peaks. Wide separation
of peaks, preferably to baseline, is desired in order to achieve maximum purification.
The speed at which any component of a mixture travels down the column in elution
mode depends on many factors. But for two substances to travel at different speeds,
and thereby be resolved, there must be substantial differences in some interaction
between the biomolecules and the chromatography matrix. Operating parameters are
adjusted to maximize the effect of this difference. In many cases, baseline separation
of the peaks can be achieved only with gradient elution and low column loadings. Thus,
two drawbacks to elution mode chromatography, especially at the preparative scale, are
operational complexity, due to gradient solvent pumping, and low throughput, due to
low column loadings. Displacement chromatography has advantages over elution
chromatography in that components are resolved into consecutive zones of pure
substances rather than “peaks”. Because the process takes advantage of the nonlinearity
of the isotherms, a larger column feed can be separated on a given column with the
purified components recovered at significantly higher concentration.
Reversed phase HPLC (RP-HPLC) has a non-polar stationary phase and an
aqueous, moderately polar mobile phase. One common stationary phase is a silica
which has been surface-modified with RMe2SiCl, where R is a straight chain alkyl
group such as C18H37 or C8H17. With such stationary phases, retention time is longer
for molecules which are less polar, while polar molecules elute more readily (early in
the analysis). An investigator can increase retention times by adding more water to the
mobile phase; thereby making the affinity of the hydrophobic analyte for the
hydrophobic stationary phase stronger relative to the now more hydrophilic mobile
phase. Similarly, an investigator can decrease retention time by adding more organic
solvent to the eluent. RP-HPLC is so commonly used that it is often incorrectly referred

26
to as "HPLC" without further specification. The pharmaceutical industry regularly
employs RP-HPLC to qualify drugs before their release.
Size-exclusion chromatography (SEC), also known as gel permeation
chromatography or gel filtration chromatography, separates particles on the basis of
molecular size (actually by a particle's Stokes radius). It is generally a low resolution
chromatography and thus it is often reserved for the final, "polishing" step of the
purification. It is also useful for determining the tertiary structure and quaternary
structure of purified proteins. SEC is used primarily for the analysis of large molecules
such as proteins or polymers. SEC works by trapping these smaller molecules in the
pores of a particle. The larger molecules simply pass by the pores as they are too large
to enter the pores. Larger molecules therefore flow through the column quicker than
smaller molecules, that is, the smaller the molecule, the longer the retention time.
This technique is widely used for the molecular weight determination of
polysaccharides. SEC is the official technique (suggested by European pharmacopeia)
for the molecular weight comparison of different commercially available low-
molecular weight heparins.
In ion-exchange chromatography (IC), retention is based on the attraction
between solute ions and charged sites bound to the stationary phase. Solute ions of the
same charge as the charged sites on the column are excluded from binding, while solute
ions of the opposite charge of the charged sites of the column are retained on the
column. Solute ions that are retained on the column can be eluted from the column by
changing the solvent conditions (e.g. increasing the ion effect of the solvent system by
increasing the salt concentration of the solution, increasing the column temperature,
changing the pH of the solvent, etc...).
Bioaffinity chromatography. This chromatographic process relies on the property
of biologically active substances to form stable, specific, and reversible complexes.
The formation of these complexes involves the participation of common molecular
forces such as the Van der Waals interaction, electrostatic interaction, dipole-dipole
interaction, hydrophobic interaction, and the hydrogen bond. An efficient, biospecific
bond is formed by a simultaneous and concerted action of several of these forces in the
complementary binding sites.

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 Aqueous normal-phase chromatography (ANP) is a chromatographic technique
which encompasses the mobile phase region between reversed-phase chromatography
(RP) and organic normal phase chromatography (ONP). This technique is used to
achieve unique selectivity for hydrophilic compounds, showing normal phase elution
using reversed-phase solvents.
Procedure

As shown in the schematic diagram in Figure above, HPLC instrumentation

includes a pump, injector, column, detector and integrator or acquisition and display
system. The heart of the system is the column where separation occurs.
1. Solvent Resorvoir: Mobile phase contents are contained in a glass resorvoir. The
mobile phase, or solvent, in HPLC is usually a mixture of polar and non-polar liquid
components whose respective concentrations are varied depending on the composition
of the sample.
2. Pump: A pump aspirates the mobile phase from the solvent resorvoir and forces it
through the system’s column and detecter. Depending on a number of factors including
column dimensions, particle size of the stationary phase, the flow rate and composition
of the mobile phase, operating pressures of up to 42000 kPa (about 6000 psi) can be
generated.
3. Sample Injector: The injector can be a single injection or an automated injection
system. An injector for an HPLC system should provide injection of the liquid sample
within the range of 0.1-100 mL of volume with high reproducibility and under high
pressure (up to 4000 psi).
4. Columns: Columns are usually made of polished stainless steel, are between 50 and
300 mm long and have an internal diameter of between 2 and 5 mm. They are
commonly filled with a stationary phase with a particle size of 3–10 µm. Columns with

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 internal diameters of less than 2 mm are often referred to as microbore columns. Ideally
the temperature of the mobile phase and the column should be kept constant during an
analysis.
5. Detector: The HPLC detector, located at the end of the column detect the analytes as
they elute from the chromatographic column. Commonly used detectors are UV-
spectroscopy, fluorescence, mass-spectrometric and electrochemical detectors.
6. Data Collection Devices: Signals from the detector may be collected on chart recorders
or electronic integrators that vary in complexity and in their ability to process, store
and reprocess chromatographic data. The computer integrates the response of the
detector to each component and places it into a chromatograph that is easy to read and
interpret.
Graph

Sample problem

In a reverse phase separation of chlorinated phenols, trichlorophenol has a retention

time of 15.0 min, when 30:70 acetonitrile-water mixture is used as the mobile phase. What
mixture of these two solvents will reduce the retention time to 12.0?

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