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LIQUID CHROMATOGRAPHY

for Clinical Applications


Wahyu Utami, M.Sc., PhD, Apt
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High Performance Liquid Chromatography


(HPLC)
• Has emerged as the most important of all chromatography which
otherwise includes TLC and GC.
• Chromatography methods are based on the separation and isolation
of the analyte of interest from all other components of a biological
sample prior to detection.
• The main advantages of chromatography methods over other
techniques are their high selectivity, sensitivity, reliability, versatility
and generally low cost of operation.
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Thin Layer Chromatography (TLC)


• TLC is used today in the clinical laboratory mostly as a screening or
semi-quantitative test for amino acids, drugs, and lipids in serum or
urine.
• TLC consists of a thin layer of sorbent such as silica gel spread
uniformly on a glass plate or plastic sheet.
• Sample is added as a small spot or band near the edge of the plate
which is then placed in a closed chamber containing the mobile
phase.
• The mobile phase migrates up the plate by capillary action and
separates the compounds in the sample which can then be visualized
by ultraviolet (UV) illumination or spraying with specific reagents.
• In its basic form, the method has serious limitations in terms of
sensitivity and selectivity as compared to modern chromatography
such as GC or HPLC and has often time been supplemented or
replaced by these techniques.
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Gas Chromatography (GC)


• GC, especially coupled to mass spectrometry (GC/MS) is used in the
clinical laboratory primarily for confirmation of drugs and screening of
amino acids.
• For example, GC/MS is commonly used in toxicology to confirm a
positive screen for opiates and opioids and can distinguish and
simultaneously report the various types, prescribed versus drug of
abuse, with high selectivity and sensitivity where an immunoassay
method cannot.
• GC consists of using a gaseous mobile phase or carrier gas to pass a
mixture of volatile solutes extracted from the sample through a
column containing the stationary phase.
• Solute separation is based on differences in vapour pressure and
interaction with the stationary phase.
• GC is best suited for the analysis of volatile organic compounds which
is also its main limitation for clinical use.
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HPLC
• HPLC is currently used for the analysis of amino acids, peptides,
proteins, carbohydrates, lipids, nucleic acids and related compounds,
vitamins, hormones, metabolites, and drugs.
• HPLC can be coupled to various detectors such as UV, fluorescence
or mass spectrometry (LC/MS and LC/MS/MS) and is routinely used
for quantitative analysis in biological samples such as blood, urine,
and other body fluids.
• HPLC consists of using a liquid mobile phase to pass under high
pressure a mixture of analytes extracted from the sample through a
column containing the stationary phase.
• Analyte separation is based on differences in interaction with both the
mobile phase and the stationary phase.
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Three of the main clinical applications of HPLC


Therapeutic drug monitoring of immunosuppressant.

Determination of vitamin D and the metabolites.

Isolation and quantification of glycosphingolipids and their metabolic


intermediates of glycolipids.
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Therapeutic drug monitoring of immunosuppressant


• Survival rates of transplant patients have increased significantly with
the introduction of immunosuppressant drugs such as cyclosporine,
sirolimus and tacrolimus.
• However, because of their complex mode of action, variable
absorption rate and narrow therapeutic index, patients need to be
monitored during treatment in order to prevent both adverse drug
reactions and organ transplant rejection events.
• The diagnostic methods used for immunosuppressant drug
monitoring are primarily based on HPLC or immunoassays.
• HPLC has specificity, sensitivity and multiplexing ability with the
simultaneous quantitation of several drugs and metabolites from a
single sample.
• HPLC with UV or mass spectrometry detection are routinely used for
therapeutic drug monitoring of immunosuppressant.
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Determination of vitamin D and the metabolites


• Vitamin D is a group of fat-soluble steroid-derived compounds that
play a role in the control of calcium and phosphorous metabolism.
• It was found to be involved in many conditions such as osteoporosis,
cancer, diabetes, CV diseases, and autoimmune diseases.
• Two forms of vitamin D are biologically active, vitamin D2 from dietary
sources and vitamin D3 which occurs naturally in human. Both forms
are metabolized in the liver and kidney to active metabolites.
• Only the measurement of 25-hydroxyvitamin D2/3 and 1,25-
dihydroxyvitamin D2/3 have proven clinical value.
• LC/MS/MS techniques have emerged as the gold standard for these
• Measurements over immunoassays and HPLC/UV because of their
ability to specifically and precisely quantify low levels of the
metabolites as well as the different forms of vitamin D, including D2
and D3 simultaneously.
AN OVERVIEW OF
LIQUID CHROMATOGRAPHY
Wahyu Utami, PhD, Apt
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FOUR BASIC LIQUID CHROMATOGRAPHY

The 4 basic liquid chromatography modes are named according


to the mechanism involved:
1. Liquid/Solid Chromatography (adsorption chromatography)
A. Normal Phase LSC
B. Reverse Phase LSC
2. Liquid/Liquid Chromatography (partition chromatography)
A. Normal Phase LLC
B. Reverse Phase LLC
3. Ion Exchange Chromatography
4. Gel Permeation Chromatography (exclusion chromatography)
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Partitioning of the sample between 2


Depend upon polarity differences phases delays or retains some
between solute molecules components more than others to effect
(based on the competition of the separation.
components of the mixture sample
for the active sites on an absorbent
such as silica gel).
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Based on the competition of Based on the size of the molecules in


different ionic compounds of the solution.
sample for the active sites on the Small molecules are able to permeate
ion-exchange resin (column- more pores and are, therefore, retained
packing). longer than large molecules.
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Types of Ion Exchange Resins


Type of Functional Exchanger Group Trade Name
Exchanger
Cation
Strong Acid Sulfonic acid (-SO3-H+) Dowex 50;
Amberlite IR 120
Weak acid Carboxyclic acid (-CO2-H+) Amberlite IRC 50

Anion
Strong base Quaternary ammonium ion (- Dowex 1;
NR3+OH-) Amberlite IRA 400
Weak base Amine group (-NH3+OH-) Dowex 3;
Amberlite IR 45
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• nRzSO3–H+ + Mn+ (RzSO3)nM + nH+

• nRzCO2–H+ + Mn+ (RzCO2)nM + nH+

• nRzNR3+OH-+ An- (RzNR3)nA + nOH-


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MECHANISM OF ION-EXCHANGE
CHROMATOGRAPHY OF AMINO ACIDS

pH2

- + +
SO 3 Na H3N
COOH

Ion-exchange Resin

- +
SO 3 H 3N
-
COO pH4.5
+
Na
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HPLC

HPLC consist of 4 principal part:


1. Mobile phase supply system
2. Sample Injection System
3. Column
4. Detector
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tr Vt 0.589tr


Resolution   
wav wav w1/ 2 av
w = peak width at the baseline between tangents drawn to the
steepest parts of the peak
w1/2 = measured at ½ the peak height
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Resolution
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Theories of
Elution Chromatography
some zone broadening
zone separation
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Rate Theory of Chromatography


Zone shapes (Gaussian curve)
Why?
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Column Efficiency
Plate height: constant of proportionality between the variance (s2)
of the band and the distance traveled (x)

Smaller plate height = narrow peaks = better separations

s2 L H: plate height (cm/plate)


H H
x N
N: number of plates
16t r2  t r2  5.5t r2
N  2   2  N
w s  w1 / 2 2 L: column length (cm)

HETP: height equivalent to a theoretical plate


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The van Deemter equation :

HETP = A + B /µ + Cµ

H=HETP=height equivalent to a theoretical plate (cm/plate)

A = eddy diffusion
B = longitudinal or molecular diffusion of the sample
components in the mobile phase or carrier gas.
C = rate of mass transfer
µ = the liquid mobile phase velocity

We want HETP to be minimum


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Eddy Diffusion Longitudinal Rate of mass


(A) diffusion (B) transfer (C)
• Due to • Due to the finite
• Due to the variety of concentration time required for the
variable-length gradients within the solute equilibrium to
pathways between column. be established
the particle in the between the two
column. • Dependent on the
gas or mobile phase phases.
• Dependent on the velocity. • Dependent on the
characteristic of the gas or mobile phase
column packing. • Function of both the
sample and the velocity.
• Can be decreased carrier gas/mobile • Is influenced by the
with smaller and phase. partition coefficient
more uniform and the relative
particles and tighter • High flow rates
reduces molecular solubility (or the
packing, which adsorb ability) of the
reduces the effective diffusions.
sample in stationary
HETP and thus • In LC, this diffusion phase.
increases efficiency. in the stationary
phase is very small • Increasing the
compared to that in solubility in mobile
GC. phase may
decrease C
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Illustration of the van Deemter equation :


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S>1: Tailing peak


S=1: Peak with
Gaussian distribution
(symmetry)
S<1: Leading peak
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HPLC column: components & specifications


 Column dimension (size): internal diameter
(0.050 to 4.6 mm) X length.
Shorter columns are generally cheaper &
generate less back pressure (low pressure it
allows the user more flexibility to adjust the
flow rate).
 Particle size (3 – 50 µm) and pore size (100-
1000Å).
The most common columns are packed with
silica particles.
Larger particles will generate less system
pressure and smaller particles will generate
more pressure. The smaller particles
generally give higher separation efficiencies.
 Stationary phase,
is generally made up of C4, C8 or C18 (RP)
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Solvents
• The reverse phase solvents are by convention installed on the
HPLC channels A and B. The A solvent by convention is the
aqueous solvent (water) and the B solvent by convention is the
organic solvent (acetonitrile, methanol, propanol).

• The A solvent is generally HPLC grade water with 0.1%


acid. The B solvent is generally an HPLC grade organic
solvent such as acetonitrile or methanol with 0.1% acid.

• The acid is used to the improve the chromatographic peak
shape and to provide a source of protons in reverse phase
LC/MS.

• The acids most commonly used are formic acid, triflouroacetic


acid, and acetic acid. A 0.1% v/v solution is made by adding
1ml of acid per liter of solvent.
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Gradient
• Start the gradient with some small % of organic (B), 2-5%
because in 100% aqueous may breakdown the column.
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Flow Rate
• Below is a table with standard flow rates for easy reference:

Sample Preparation
• The sample is normally reconstituted in the A solvent to
maximize binding to the column.
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Optimizing the Separation


• To improve the separation or to shorten the run time.

• Shorter analysis times are always better for work efficiency.

• The first step in the optimization is to determine the %B at


which the last peak elutes.

• Remember that all HPLC systems have a gradient delay (the


time between when the software tells the pumps to start
pumping at a certain mobile phase composition and the time it
takes for that solvent composition to reach the column and
have an effect).
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Example:

This would make the gradient shallower


and possibly give a better peak
separation.

To shorten the run time one could


rewrite the gradient to look like this:
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What is HPLC Equilibration?


• The column must be equilibrated, re-
equilibrated to the initial high aqueous
solvent composition before another
analysis can be performed.
• The equilibration time depends on the
column length, flow rate and the
hydrophobicity of your peptides. Some
chromatographers use 10 minutes as
their standard equilibration time.
• You should determine the equilibration
time experimentally, the criteria will be:
does my analyte really stick to the
column and chromatograph
appropriately and reproducibly with
subsequent analyses?
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Should I Control Column Temperature?


• Many HPLCs provide the option to control column
compartment temperature. If your HPLC does not have this
capability a heated column jacket can be purchased from many
suppliers.
• The most common running temperature is 40°C, this places the
column compartment well above even the most extreme
ambient temperature fluctuations.
• In addition to maintaining constant temperature, temperature
can be used to influence the chromatographic separation
• Higher temperature is better, peaks will be sharper and elute
earlier.
• It is not too uncommon to perform chromatography at 60°C and
some daredevils even go to 80°C. Remember though that
higher temperature will lead to a shorter column lifetime and
some columns may not be able to tolerate 60°C.
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Preparing for the First Run of the Day


• One observation is that if you start up a reverse phase analysis
from a dead stop with a column that has perhaps been sitting in
high aqueous conditions for up to 10 hours the analysis will
give irreproducible results.
• Conventional wisdom has it, you want to first flush the column
with the highest % organic of your method for at least 3 column
volumes and then bring it back to the equilibrating condition.
This practice may have the advantage of getting you to
standard equilibration conditions faster and it will also clean
your column.
• A better alternative is to make the first run a blank run (or
"preparation run") and then the next run can be your real
analysis.
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After the Last Run of the Day


• We store our columns in 50/50 methanol/water without any
acid.
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Selecting a mobile phase


Mobile phases of Solvent Polarity Solvent Polarity
intermediate Index (P’) Index (P’)
polarity can be n-hexane 0.1 ethyl acetate 4.4
fashioned by mixing
together two or more cyclohexane -0.2 iso -propanol 3.9
of the mobile phases
in the table. carbon 1.6 chloroform 4.1
tetrachloride

P’AB= ΦAP’A+ ΦBP’B toluene 2.4 acetone 5.1


benzene 2.7 ethanol 4.3
P’A and P’B are the methylene 3.1 acetonitrile 5.8
polarity indexes for chloride
solvents A and B, and
n-propanol 4.0 methanol 5.1
ΦA and ΦB are the
volume fractions of the Tetrahydrofuran 4.0 water 10.2
two solvents.
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Example:
• Reverse-phase HPLC separation is carried out using a
mobile-phase mixture of 60% v/v water and 40% v/v
methanol. What is the mobile phase’s polarity index?

• Solution:
From the table we find that the polarity index is 10.2 for
water and 5.1 for methanol. Using the equation 1, the
polarity index for a 60:40 water–methanol mixture is
P’AB = (0.60)(10.2) + (0.40)(5.1) = 8.2
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Isocratic vs Gradient Elution


• When a separation uses a single mobile phase of fixed
composition it is called an isocratic elution.
• A separation in which the mobile phase composition is
changed during the separation process is described as a
gradient elution.
• For a reverse-phase separation the initial mobile-phase
composition is relatively polar. As the separation
progresses, the mobile phase’s composition is made less
polar.
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Isocratic or Gradient?
• You have been given a C18 column, an unknown sample and
been asked to develop a reversed phase HPLC method.
• Where do you start?
• Do you use isocratic or gradient elution?
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Step 1. Run a scouting gradient


• This allows you to investigate the elution behaviour of the
analytes within the sample.
• Typically we use a 5 – 100% gradient over around 20 minutes
and examine the results to make some predictions regarding
the optimum mobile phase composition.
• Typically it is not strictly necessary to control pH for neutral
species; however, at lower mobile phase pH peak shape for
polar compounds will improve.
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Step 2.
Isocratic analysis is
possible when Δt < 0.25 tg

If Δt > 0.25 tg,


use gradient elution

where: Δt =(tf – ti)

tg = gradient time
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Factors affecting retention time:

• length of column
• packing material

• type of carrier gas/mobile phase


• flow rate of carrier gas/mobile phase
• temperature of column.
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Detectors
• Refractive Index (RI) “universal detector”
Measures changes in refractive index of the eluent that results from the
presence of solutes as they emerge from the column.
It cannot used effectively with gradient elution due to a change in the baseline
It’s very sensitive to temperature changes.
• UV-Vis much better sensitivity than RI.
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Detectors
• Diode Array (DAD)
Detects the absorption in UV to VIS region. While a UV-VIS detector has only
one sample-side light-receiving section, a DAD has multiple photodiode
arrays to obtain information over a wide range of wavelengths at one time.
DADs differ from UV-VIS detectors in that light from the lamps is shone
directly onto the flow cell, light that passes through the flow cell is dispersed
by the diffraction grating, and the amount of the dispersed light is estimated
for each wavelength in the photodiode arrays.
Compared with a UV-VIS detector, the DAD has the following disadvantages:
noise is large because the amount of light is small; the DAD is also
susceptible to various changes, such as lamp fluctuations, because the
reference light cannot be received. However, the DAD has recently been
improved to reduce its difference in performance from UV-VIS detectors.
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Detectors
• Fluorescence
Improved selectivity over UV-Vis.
• Electrochemical (EC)
• amperometric
• Mass spectrometry (MS)
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Properties of a Good Detector


• High sensitivity - Response/Concentrations

• Universal or selective response


• selectivity - ability to distinguish between species

• Rapid response

• Linearity - concentration range over which signal proportional


to concentration

• Stability with respect to noise (baseline noise) and time (drift)


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Applications of Chromatography
• Qualitative Analysis
• Quantitative Analysis
• Analyses Based on Peak Height
• Analyses Based on Peak Areas
• Calibration and Standards
• The Internal Standard Method
• The Area Normalization Method