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Types of HPLC
Based on the chemical nature of the stationary phase, and on the retention mechanism, HPLC
can be divided into three types, which cover almost 90% of all chromatographic applications.
Figure 1. Chemical modification of the silica gel using octadecyl ligands (C18).
Flasks for the mobile phase storage: Using high pressure pumps to deliver liquid, the
solvent have to be gas-free in HPLC experiments. The excess gas in the mobile phase causes
several problems during the analysis. Because of the compressibility of the gases, the pump
pressure and the flow rate will fluctuate, and it would cause significant disturbance in the
detection and in the repeatability of the chromatographic data. Because of these reason, the
mobile phase has to be degassed. Degassing may be accomplished by one of the following
methods or their combination:
Degassing the liquid under vacuum-heavy-walled flask is really important, in this
case.
Heating the liquid until its boiling occur.
Placing the container of liquid in an ultrasonic bath, or inserting an ultrasonic probe in
it.
Bubbling a fine stream of He through the liquid; He has the unique ability to purge
other gases out of solutions.
Some instruments are equipped with built-in degasser, but its capacity is not enough in
every case.
Pumps: The pumps applied in the practice of HPLC have to provide a constant and almost
pulse-free flow of the mobile phase through the system. If we use one pump, the composition
of the mobile phase is constant during the experiment, which is called isocratic elution. With
the aid of two or more pumps, using a time program, the composition of the mobile phase is
variable. When the mobile phase composition is changing during the analysis, the technique is
called gradient elution.
Injector: Injector for liquid chromatographic system should provide the possibility of
injecting the liquid sample within a large volume range with high reproducibility and under
high pressure (up to 1200 bar). They should also produce minimum band broadening and
minimize possible flow disturbances. Generally, the most useful and widely used sampling
device for modern LC is the six-port Rheodyne valve.
Figure 3. Manual six-port injector with the two position for sample introduction.
In the six-port Rheodyne valve the sample is introduced into the sample loop (in LOAD
position) using a special syringe. A clockwise rotation of the valve rotor (INJECT position)
places the sample-filled loop into the mobile-phase stream, with subsequent injection of the
sample onto the top of the column through a low-volume, cleanly swept channel.
Columns: The part of the HPLC where the stationary phase is immobilised and the retentions
of the compounds take place. Modern HPLC column beds used in adsorption chromatography
are small rigid porous particles with high surface area.
Detectors: Detectors equipped with the flow-through cell were a major breakthrough in the
development of modern liquid chromatography. Such detection was first applied by the group
of Tiselius, in Sweden in 1940, by continuously measuring the refractive index of the column
effluent. Current LC detectors have wide dynamic range, and have high sensitivities often
allowing the detection of nanograms of material. A few types of detectors:
UV/Vis detector
Fluorescence detector
Conductivity detector
Refractive index detector
Mass spectrometer (MS)
In the last decade there is a significant progress in the development of LC-MS interfacing
systems. MS as an on-line HPLC detector is said to be the most sensitive, selective and in the
same time the most universal detector. But it is still the most expensive one.
Time (min)
Retention volume (VR): is a more exact and global retention parameter. It is the product of
the retention time and the volumetric flow rate of the mobile phase. Represents the volume of
the mobile phase passed through the column while eluting a particular component.
Retention volume is independent of the flow parameters of the particular run, but it depends
on the geometrical parameters of the column.
Retention factor (k’) is dimensionless and independent of any geometrical parameters of the
column or HPLC system. It could be considered to be a thermodynamic characteristic of the
adsorbent-compound-eluent system.
Theoretical plate number (N): The plate model supposes that the chromatographic column
contains a large number of separate layers called theoretical plates. Separate equilibrations of
the sample between the stationary and mobile phase occur in these ”plates”. The analyte
moves down the column by transfer of equilibrated mobile phase from one plate to the next. It
is important to remember that these theoretical plates do not really exist; they are a figment
of the imagination that help us to understand the processes at work in the column. They also
serve as a way of measuring column efficiency, either by starting the number of theoretical
plates in a column.
Theoretical plate height (H):
Instrumentation:
Chemicals (samples):
Figure 6. Chemical structures of the two studied compounds; caffeine (a) and paracetamol (b).
Tasks:
CH3OH % paracetamol
CH3OH % caffeine
Calibration curve and quantitative analysis: Dilute three times the standard
stock solution and measure the peak area of the standards (three times each
concentration). Calculate the average peak area (and standard deviation) of the
three injections. Construct a calibration curve of peak area versus
concentration. Inject the ten-fold diluted Saridon tablet solution three times and
determine the concentration of caffeine and paracetamol based on the average
of the measured peak areas and the constructed calibration curve. Calculate the
concentration (mg/tablet) of the pharmaceutically active compounds in a single
Saridon tablet.
1.6 Questions
1.2 Introduction
1.3 Theory
The mobility, for a given ion and medium, is a constant which is characteristic of that ion.
The mobility is determined by the electric force that the molecule experiences, balanced by its
frictional drag through the medium.
The electric force can be given by
𝐹𝑒 = 𝑞 ∙ 𝐸
𝐹𝑓 = - 6 π η r v
qE = 6πηrv
where q = ion charge
η = solution viscosity
r = ion radius
v = ion velocity
During electrophoresis a steady state, defined by the balance of these forces, is attained. At
this point the forces are equal but opposite and
Fe = Ff
qE = 6πηrv
Solving for velocity and substituting equation (4) into equation (1) yields an equation that
describes the mobility in terms of physical parameters
𝑞
𝜇𝑒 =
6𝜋𝜂𝑟
From this equation it is evident that small, highly charged species have high mobilities
whereas large, minimally charged species have low mobilities.
Electro-osmotic flow (EOF)
EOF is the bulk flow of liquid in the capillary and is a consequence of the surface
charge on the interior capillary wall.
The EOF results from the effect of the applied electric field on the solution double-
layer at the wall (Figure 2). The potential difference created close to the wall is known as the
zeta potential.
Under aqueous conditions most solid surfaces possess an excess of negative charges.
EOF is most strongly controlled by the numerous silanol groups (SiOH) that can exist in
anionic form (SiO-) (Figure 3a). Although the exact pI of fused silica is difficult to determine,
EOF becomes significant above pH 2.5. Counterions (cations), which build up near the
surface to maintain charge balance, form the doublelayer (Figure 3b). When the voltage is
applied across the capillary the cations forming the diffuse double-layer are attracted toward
the cathode. Because they are solvated their movement drags the bulk solution in the capillary
toward the cathode. This process is shown in schematic form in Figure 3c.
The magnitude of the EOF can be expressed in terms of mobility by
𝜁𝜀𝜀0
𝜇𝐸𝑂𝐹 = −
4𝜋𝜂
where
The zeta potential is essentially determined by the surface charge on the capillary wall. Since
this charge is strongly pH dependent, the magnitude of the EOF varies with pH. At high pH,
where the silanol groups are predominantly deprotonated, the EOF is significantly greater
than at low pH where they become protonated. The zeta potential is also dependent on the
ionic strength of the buffer. Increased ionic strength results in double-layer compression,
decreased zeta potential, and reduced EOF.
The EOF controls the amount of time solutes remain in the capillary by superposition
of flow on to solute mobility. Thus, EOF causes movement of nearly all species, regardless of
charge, in the same direction. Under normal conditions (that is, negatively charged capillary
surface), the flow is from the anode to the cathode.
As depicted in Figure 4, cations migrate fastest since the electrophoretic attraction
towards the cathode and the EOF are in the same direction, neutrals are all carried at the
velocity of the EOF but are not separated from each other, and anions migrate slowest since
they are attracted to the anode but are still carried by the EOF toward the cathode (since the
magnitude of the flow can be more than an order of magnitude greater than their
electrophoretic mobilities).
Modification of capillary wall charge can decrease EOF (even to zero, if the capillary is
covalently coated with a polymer). In these circumstances, anions and cations can migrate
in opposite directions.
A unique feature of EOF in the capillary is the flat profile of the flow, as depicted in
Figure 5. Since the driving force of the flow is uniformly distributed along the capillary there
is no pressure drop within the capillary, and the flow is nearly uniform throughout. The flat
flow profile is beneficial since it does not contribute to the dispersion of solute zones. Peak
5
efficiency, often in excess of 10 theoretical plates, is due in part to the plug profile of the
electro-osmotic flow.
Modes of operation
CZE is the simplest form of CE, mainly because the capillary is only filled with buffer.
Separation occurs because solutes migrate in discrete zones and at different velocities, based
on their charge to size ratio. Separation of both anionic and cationic solutes is possible due to
electro-osmotic flow (EOF). Neutral solutes do not migrate and all co-elute with the EOF.
Capillary. Ideal properties of the capillary material include being chemically and electrically
inert, UV-Visible transparent flexible and robust, and inexpensive. Meeting most of these
requirements, fused silica is the primary material employed today. Effective control of
capillary temperature is important for reproducible operation.
Injection. The two most common sample injection methods are hydrodynamic (by application
of pressure) and electrokinetic (by application of low field strength).
Detection. UV-Visible absorption is the most widely used detection method. An optical
window in the capillary is easily created by removal of a small (1-3 mm) section of the
protective polyimide coating. Other detectors are LIF (laser induced fluorescence),
conductivity detector, mass spectrometer, etc.
Electropherogram
30
MeHg-CYS
Hg(CYS)2
mAU
PhHg-CYS
CYS
20
EtHg-CYS
10
0
3 6 9 min 12
The time required for a solute to migrate to the point of detection is called the “migration
time”, and is given by the quotient of migration distance and velocity. The migration time and
other experimental parameters can be used to calculate the apparent solute mobility using
𝑙 ∙𝐿
𝜇= [𝑐𝑚2 / V ∙ s]
𝑡∙𝑈
where
𝜇𝑎 = 𝜇𝑒 + 𝜇𝐸𝑂𝐹
U = applied voltage
l = effective capillary length (from injection to the detector)
L = total capillary length
t = migration time
In the presence of EOF, the measured mobility is called the apparent mobility, µa. The
effective mobility, µe, can be extracted from apparent mobility by independently measuring
the EOF using a neutral marker (e.g. acetone) that moves at a velocity equal to the EOF.
Efficiency
𝑡 2
𝑁 = 16 ( )
𝑤
where
t = migration time
𝑤1/2 = temporal peak width at half height
Theoretical plate number can be related to the HEPT (height equivalent to a theoretical
plate), H, by
𝑙
𝐻=
𝑁
where
l = effective capillary length
Resolution (R)
Resolution of sample components is the ultimate goal in separation science. Resolution is
most simply defined as
𝑅 = 2 (𝑡2 − 𝑡1 )/(𝑤1 + 𝑤2 )
where
t = migration time
w = baseline peak width (in time)
Turn on the computer, capillary electrophoresis instrument, and start the program. Put the
capillary cassette with the capillary in the instrument.
Running conditions:
-Instrument:
-Buffer:
-Capillary length (total and effective):
-Injection mode:
-Temperature:
-Voltage:
-Polarity:
-Detection wavelength:
Samples:
Benzoic acid, sorbic acid and ascorbic acid (vitamin C) of known concentration (standard
solutions)
Lime juice.
O OH
HO O O
OH
H3C OH
O HO OH
Tasks
1. Make the electrophoretic run of a mixture of standards.
2. Identify the peaks (compounds) according to their charge/mass ratio.
3. Determine t (migration time) and w1/2 (peak width at half height) of the peaks.
4. Calculate µa (apparent mobility) of each compound.
5. Determine separation efficiency by the calculation of parameters N, H and R.
6. Make the electrophoretic run of lime juice.
7. Identify the peaks (qualitative analysis) and calculate the concentration of compounds
(quantitative analysis) by comparing the electropherogram to that of standard mixture.
1.7 Bibliography
1.1 Aim
Overview of the main parts of GC, the setting of the measuring method used in the
practice, manual injection of Sinupret sample and standard samples, qualitative and
quantitative analysis of ethanol in Sinupret sample by flame ionization detection, examination
of factors affecting gas chromatographic separation.
1.2 Introduction
1.3 Theory
Basic terms
Retention
The analyte has been retained because it spends a time (tS) in the stationary phase. Separation
is based on the different times that components spend in the stationary phase.
Minimum possible or dead time (tM) [min] is the time it takes for an unretained species to
pass through a chromatographic column.
Retention time (tR) [min] is the easiest way to define chromatographic retention. We
measure the time between the injection and the maximum detector respond (peak maximum)
for the correspondent compound:
tR = tS + tM
Adjusted retention time (tR’) [min] for a retained solute is the additional time required to
travel the length of the column, beyond the time required by unretained solvent:
tR’= tR- tM
Retention factor (k’) is dimensionless and independent of any geometrical parameters of the
column. It could be considered to be a thermodynamic characteristic of the adsorbent-
compound-eluent system:
t t
k’= R M
tM
Relative retention (Selectivity factor) (α):
=k’2/k’1
where k’2 > k’1, so
The greater the relative retention the greater the separation between two components.
Efficiency of Separation: Two factors contribute to how well compounds are separated by
chromatography. One is the difference in elution times between peaks: the farther apart, the
better their separation. The other factor is how broad the peaks are: the wider the peaks, the
poorer their separation.
Resolution (R) of a column tells us how far apart two bands are relative to their widths. The
resolution provides a quantitative measure of the ability of the column to separate two
analytes. In chromatography, the resolution of two peaks from each other is defined as:
t R 2 t R1
R= 2 *
w1 w2
where w is the peak width at baseline.
For quantitative analysis, a resolution > 1.5 is highly desirable.
Number of theoretical plate (N): The plate model supposes that the chromatographic
column is contains a large number of separate layers, called theoretical plates. Separate
equilibrations of the sample between the stationary and mobile phase occur in these "plates".
The analyte moves down the column by transfer of equilibrated mobile phase from one plate
to the next. It is important to remember that the plates do not really exist; they are a figment
of the imagination that helps us understand the processes at work in the column. They also
serve as a way of measuring column efficiency, either by stating the number of theoretical
plates in a column.
2
t
N=16* R
w
where w is the peak width at baseline.
Plate height / Height Equivalent to a Theoretical Plate (H) is approximately the length of
column required for one equilibration of solute between mobile and stationary phases:
H=L/N
where L is length of the column.
The chromatogram is a graph showing the detector response as a function of elution time. It
has two main parameters: the retention time (tR) and the peak area. The retention time is used
for the qualitative analysis of components. Quantitative analysis is based on the area of a
peak. In the linear response concentration range, the area of a peak is proportional to the
quantity of that component.
In gas chromatographic analysis an internal standard (IS) is generally added to the sample.
Internal standards are similar in analytical behavior to the compounds of interest, and not
expected to be found in the samples. Spiking the samples with internal standards helps to
compensate imprecisions derived from sample preparation or variable injection volumes. A
constant amount of the internal standards added to all samples, and that same amount of the
internal standard is also included in each of the calibration standards. The ratio of the peak
area of the target compound in the sample to the peak area of the internal standard in the
sample is compared to a similar ratio derived for each calibration standard. This ratio is
termed the response factor (RF) or relative response factor (RRF), indicating that peak area of
the target compound is calculated relative to that of the internal standard:
Ax ∗ CIS
𝑅𝐹 =
AIS ∗ Cx
Ax-area of the compound
cx- concentration of the compound
AIS-area of the internal standard
cIS- concentration of the internal standard
Ax /AIS
(Ax3/AIS)3
(Ax2/AIS)2
(Ax1/AIS)1
Cx /CIS
(Cx1/CIS)1 (Cx2 /CIS)2 (Cx3 /CIS)i
(Ax2/AIS)2
Figure 2. Calibration in internal standard method
(Ax1/AIS)1
Gas Systems
The mobile phase or carrier gas is helium. The choice of gases is often dictated by the
type of detector used. The gases of flame ionization detector are hydrogen and air.
Injector
The split / splitless injector (Figure 3.) is used in conjunction with capillary column
GC. Injection takes place into a heated glass or quartz liner rather than directly onto the
column.
In the split mode, the sample is split into two unequal portions the smaller of which
goes onto the column. This technique is used with concentrated samples.
In the splitless mode, the entire sample is introduced onto the column.
Two general types of columns are encountered in gas chromatography, packed and
capillary. Capillary columns (Figure 4.) are made from fused silica, usually coated on the
outside with polyimide to give the column flexibility. The wall of the column is coated with
the liquid stationary phase. The most common type of coating is based on organo silicone
polymers, which are chemically bonded to the silanol groups on the wall of the column and
the chains of the polymers are further cross-linked. Polyethylene glycol is also commonly
used as a polar stationary phase for gas chromatography (wax-column).
Important separation factors are the length and internal diameter of column, the type
and thickness of stationary phase.
The column is ordinarily house in a thermostated oven. The optimum column
temperature depends upon the boiling point of the sample and the degree of separation
required. The ovens can be programmed to either produce a constant temperature, isothermal
conditions or a gradual increase in temperature.
Detector
Detectors used in GC vary in nature depending upon the characteristics of the analyte
and the circumstances of its determination.
The flame ionization detector (Figure 5.) is the most widely used and generally
applicable detector for gas chromatography. The flame is mixed with hydrogen and air. When
solute molecules contained in the carrier gas elute from the column and pass into the detector
they burn in the flame and in doing so, generate ions which move to the collector electrode,
due to the potential difference between the jet and the electrode. The resulting ionization
current is amplified and fed to the data system.
- Pipette 200 L of the Sinupret sample into a 10 mL volumetric flask. Add 40 μL internal
standard (n-propanol) to the sample and fill to the mark with water!
- Set the parameters on the gas chromatograph with the help of the instructor!
- Set the temperature of split-splitless injector! T=200C
- Set the temperature of FID detector! T=225C
- Set the temperature of oven!
1. We start the measurement on 50C, which is hold for 0,5 min, than we start
to increase the temperature with 25C/min to 150C.
2. We start the measurement on 50C, which is hold for 6,5 min.
Procedure 3. Determination of dead time
Standard for dead time determination: steam of dichloromethane
How to inject?
Rinse the syringe 5 times with solvent (distilled water) and take up 1 L of sample and 1 L of air and push the
needle through the rubber septum into the heated injection port of the chromatograph. Push the sample and
remove the syringe then start the measuring method with start button! Rinse the syringe with solvent before
every injection!
- Inject 1 l of the Sinupret sample into the gas chromatograph! Record the retention time
of components!
tR (min) in sample
1. component
2. component
- Inject 1 l of standard ethanol solution into the gas chromatograph! Record the retention
time of ethanol!
- Inject 1 l of the internal standard solution (n-propanol) into the gas chromatograph! Record
the retention time of n-propanol!
tR (min) in standard
solution
ethanol
n-propanol
In the notebook identify the ethanol peak on the chromatogram of Sinupret sample! (Compare
the retention times of the components in the Sinupret sample with the retention times in the
standard solutions)!
Procedure 5. Quantitative analysis-Determination the amount of ethanol in
Sinupret sample
In the notebook construct the calibration curve! Give the ethanol content of Sinupret sample
in v/v%!
A et A IS
Sinupret sample
-Change the oven temperature from programmed to isothermal! T= 50C (6,5 min)!
-Inject the Sinupret sample into the gas chromatograph! Record the retention time of
components!
tR (min) in sample
1. component
2. component
In the notebook compare the recorded chromatogram with the chromatogram recorded
with temperature program:50C (0,5 min)25C /min 150C (0 min)!
In the notebook calculate the k,R, N, H, R parameters with the help of table below
and the chromatogram recorded under the practice!
(Use the chromatogram of the Sinupret sample recording with programmed temperature)
Name of the Calculation ethanol n-propanol
parameter
tM
tR
tR’
k’
w
N
H
R
1.6 Questions
1.7 Bibliography