HIGH PERFORMANCE

LIQUID
CHROMATOGRAPHY
(HPLC/KCKT)

Yuli Ainun Najih

High
Pressure
L iquid
Chromatograph
y
High
Performance
L iquid
Chromatograph
y
High
Priced
L iquid
Chromatograph
y
 Introduction
HPLC is a form of liquid chromatography used to
separate compounds that are dissolved in solution.
HPLC instruments consist of a reservoir of mobile
phase, a pump, an injector, a separation column,
and a detector.

Compounds are separated by injecting a sample

mixture onto the column. The different component
in the mixture pass through the column at
differentiates due to differences in their partition
behavior between the mobile phase and the
stationary phase. The mobile phase must be
degassed to eliminate the formation of air bubbles.
 What is HPLC?
Originally referred to as High-Pressure
Liquid Chromatography

Now more commonly called High

Performance Liquid Chromatography

HPLC is really the automation of traditional

liquid chromatography under conditions
which provide for enhanced separations
during shorter periods of time, utilizing
very small particles, small column
diameters, and very high fluid pressures.
INSTRUMENTATION
HPLC system
 HPLC Chromatograph
injectors
The function of the injector is to place the sample
into the high-pressure flow in as narrow volume as
possible so that the sample enters the column as a
homogeneous, low-volume plug. To minimize
spreading of the injected volume during transport to
the column, the shortest possible length of tubing
should be used from the injector to the column.

When an injection is started, an air actuator rotates

the valve: solvent goes directly to the column; and
the injector needle is connected to the syringe. The
air pressure lifts the needle and the vial is moved
into position beneath the needle. Then, the needle
is lowered to the vial.
 HPLC columns
The column is one of the
most important
components of the HPLC
chromatograph because
the separation of the
sample components is
achieved when those
components pass through
the column. The High
performance liquid
chromatography apparatus
is made out of stainless
steel tubes with a diameter
of 3 to 5mm and a length
ranging from 10 to 30cm.
Normally, columns are filled
with silica gel because its
particle shape, surface
properties, and pore structure
help to get a good separation.
Silica is wetted by nearly
every potential mobile phase,
is inert to most compounds
and has a high surface activity
which can be modified easily
with water and other agents.
Silica can be used to separate
a wide variety of chemical
compounds, and its
chromatographic behavior is
generally predictable and
reproducible.
KOLOM HPLC
 HPLC Column

solvent
(mobile phase) to detector
and
sample
wax coated beads

nonpolar
stationary phase

microscopic view of bead

3 m
Chromatography Stationary
Phases
Silica Gel Derivatized Silica Gel

Where R = C18H37
hydrocarbon chain
(octadecylsilyl deriv.
silica or C18)

bulk (SiO2)x surface bulk (SiO2)x surface

relatively polar surface relatively nonpolar surface

normal phase reversed phase
Several column types
(can be classified as )
Normal phase
Reverse phase
Size exclusion
Ion exchange
 HPLC - Modes
Normal Phase.
- Polar stationary phase and non-polar
solvent.

Reverse Phase.
- Non-polar stationary phase and a polar
solvent.
 Normal Phase / Reversed
Phase
Stationary
Mobile phase
phase
Normal High polarity Low polarity
phase (hydrophilic) (hydrophobic)

Reversed Low polarity High polarity

phase (hydrophobic) (hydrophilic)

17
Types of Partition Chromatography

Normal-Phase Reversed-Phase

Non-polar mobile phase

OH CH3

Polar mobile phase

OH CH3 Non

No-Polar stationary phase

Polar stationary phase

Highly polar
polar
OH CH3

OH Moder- CH3 Moder-

ately
ately
OH polar CH3 polar

OH CH3
Non Highly
polar
OH polar CH3
OH CH3
 Reversed Phase
Chromatography
Stationary phase: Low polarity
Octadecyl group-bonded silical gel
(ODS)
Mobile phase: High polarity
Water, methanol, acetonitrile
Salt is sometimes added.

19
 Separation Column for
Reversed Phase
Chromatography
C18 (ODS) type Phenyl type
C8 (octyl) type TMS type
C4 (butyl) type Cyano type

CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2

Si -O-Si
CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3

C18 (ODS)

20
Effect of Chain Length of
Stationary Phase
C8

Medium
C18 (ODS)

Strong C4

Weak

21
 Relationship Between
Retention Time and Polarity

C18 (ODS) OH

Weak
Strong
CH3

22
Relationship between Polarity of Eluent
and Retention Time in Reversed Phase
Mode
Eluent: Methanol / Water

60/40

70/30

80/20
23
 Common Reverse Phase
Solvents
Methanol CH3OH

CH3CN
Acetonitrile
Tetrahydrofuran

Water H2O
 Guidelines for Setting Mobile Phase
Conditions (1)
Neutral (Nonionic) Substances
Eluent Composition
Water / acetonitrile
Water / methanol
Separation Adjustment
Changing the mixing ratio of the water
and organic solvent
Changing the type of organic solvent

25
 Bonded Phases
C-2 Ethyl Silyl -Si-CH2-CH3

C-8 Octyl Silyl -Si-(CH2)7-CH3

C-18 Octadecyl Silyl -Si-(CH2)17-CH3

CN Cyanopropyl Silyl -Si-(CH2)3-CN

 Normal phase

In this column type, the retention

is governed by the interaction of
the polar parts of the stationary
phase and solute. For retention to
occur in normal phase, the
packing must be more polar than
the mobile phase with respect to
the sample
 Reverse phase
In this column the packing material is
relatively nonpolar and the solvent is polar
with respect to the sample. Retention is the
result of the interaction of the nonpolar
components of the solutes and the nonpolar
stationary phase. Typical stationary phases
are nonpolar hydrocarbons, waxy liquids, or
bonded hydrocarbons (such as C18, C8, etc.)
and the solvents are polar aqueous-organic
mixtures such as methanol-water or
acetonitrile-water.
 Size exclusion
In size exclusion the HPLC column is
consisted of substances which have
controlled pore sizes and is able to be
filtered in an ordinarily phase according
to its molecular size. Small molecules
penetrate into the pores within the
packing while larger molecules only
partially penetrate the pores. The large
molecules elute before the smaller
molecules.
 Ion exchange
In this column type the sample
components are separated based upon
attractive ionic forces between molecules
carrying charged groups of opposite
charge to those charges on the stationary
phase. Separations are made between a
polar mobile liquid, usually water
containing salts or small amounts of
alcohols, and a stationary phase
containing either acidic or basic fixed
sites.
 Stationary Phases
Polar (Normal Phase):
Silica, alumina

Non-Polar (Reversed Phase)

ODS Silica gel
C18, C8
 The Mobile Phase

Normal chromatography
Hexane ; dichloromethane; isopropanol; methanol

Increasing strength

Reverse phase chromatography

water ; methanol; acetonitrile; tetrahydrofuran
(THF)
Increasing strength
 Solvent Reservoir
Mobile phase
isocratic elution - single solvent
separation teachnique
gradient elution - 2 or more solvents,
varied during separation
To carry sample into the column
 Mixing, Filtration, and
Offline Degassing of the
Eluent
Decompression Membrane filter with pore
by aspirator size of approx. 0.45 m

Decompression
by aspirator

Ultrasonic
cleaning unit

34
 Polarity of Substances
Polarity Miscibility of
Property of a substance solvents
whereby the positions of Solvents of similar
the electrons give rise to polarities can be easily
positive and negative dissolved together.
poles
Polar and nonpolar
Water: Polar
molecules have a
Methane: Nonpolar similar relationship to
H that ofHwater Oand oil.

O H C C
C O

H H H
H H +
H Acetic acid
Methane Water

35
 Nonpolar (Hydrophobic) Functional Groups
and Polar (Hydrophilic) Functional Groups

Nonpolar Polar Functional

Functional Groups Groups
-(CH2)nCH3 -COOH
Alkyl groups Carboxyl groups
-C6H5 -NH2
Phenyl groups Amino groups
-OH
Hydroxyl groups

36
High Performance yang
didefinisikan oleh Prof.
Horvart
 Components of HPLC
1. Solvent Reservoir
2. Pumps
3. Sample Injection System
4. Columns
5. Detectors
6. Data Processing
7. Waste
INTRUMENSTATION

solvent

pump

injector

column

detector
INJECTOR/RHEODYNE

41
SAMPLE INJECTION SYSTEM:
Several devices are available either for manual or auto injection of the
sample. Different devices are: 1.Septum injectors
2. Stop flow injectors
3. Rheodyne injectors (loop valve type)
Rheodyne injector is the most popular injector. This has a fixed volume loop like 20
l or 50 l or more. Injector has 2 modes. Load position and Inject mode.

Limit of precision of HPLC

Sample size: 0.5 ~ 500 L
No interference with the pressure
Based on a sample loop, 1 ~ 100 L, Reproducibility: 0.1%, P < 7000 psi
Auto sampler: inject continuously variable volume 1 L 1 mL
Controlled temperature environment for derivatization reaction.
 Filtration and Centrifugal
Separation
In general, filter every
sample before injection!
It is convenient to use a
disposable filter with a
pore diameter of
approx. 0.45 m.
Centrifugal separation is
applicable for samples
that are difficult to filter.
Filter Syringe

44
HPLC Column
Guard Column and Pre-
column
Guard column

Pre-column

46
 Replacement of Eluent
Mutually insoluble solvents
must not be exchanged
directly.
Aqueous solutions
containing salt and organic
solvents must not be
exchanged directly.

Water Buffer solution

2-Propanol Water

Water-soluble
Hexane
organic solvent
47
HPLC Pump
 Pumps

To produce an appropriate pressure

to push solvent into the sample.

A pump capable of pumping solvent

up to a pressure of 4000 psi and at
flows of up to 10 ml/min
HPLC Autosampler and Injector
Sample Injection System
sample valve
Syringe/injector

Syringe :
manual
Autoinjector

A fixed-volume loop of between 1

200 l (20 l is often used as standard)
HPLC Detector UV/Visible
Spectrophotometer
 HPLC Detectors

UV/Vis
Refractive index
Fluorescence
Evaporative light scattering
(ELSD)
MS
Diode Array Detector (DAD)
Photo diode array detector
Typical Diode Array Signal Output
 Comparison of Detectors
Possibility of
Selectivity Sensitivity
Gradient System
Light-absorbing
Absorbance ng Possible
substances

Fluorescence Fluorescent substances pg Possible

Differential
None g Impossible
refractive index
Evaporative light
Nonvolatile substances g Possible
scattering
Electrical
Ionic substances ng Partially possible
conductivity
Oxidizing / reducing
Electrochemical pg Partially possible
substances
Note: The above table indicates general characteristics. There are exceptions.

56
HPLC Waste Collection
 Data Processing
Using specific sowtare that is connected to
HPLC machine
Receive the information from HPLC
machine and present it as a graph
The graph describes about qualitative data
(Retention time) and quantitative data
(area under curve)
 Varian HPLC
System
9060 Polychrom Computer
(Diode Array) Workstation
Detector

9050 Variable
9010 Solvent UV/Vis Detector
HPLC Solvent Delivery System
Reservoirs

Rheody
ne
Injector
Keep an eye on
HPLC
these 4 screens!
Colum
n
Strength of detector signal
Retention Factor, k

tR t R t0
k
t0
t0
tR: Retention time
t0: Non-retention time

Time

62
Theoretical Plate Number, N

2
tR
N 16
W
2
tR
5.54
H W1/ 2
W1/2 2
H1/2 tR H
2
W Area
63
Evaluation of Column Efficiency Based on
Theoretical Plate Number

If the retention times If the peak width is the

are the same, the peak same, the retention
width is smaller for the time is longer for the
one with the larger one with the larger
theoretical plate theoretical plate
number. number.
N: Large N: Small
N: Large

N: Small

64
 Separation Factor, a
Separation factor: Ratio of ks of two
peaks

k1 k2 k2

k1
(k 2 k1 )

65
 Resolution, RS

tR1 tR2

t R 2 t R1
RS
1
(W1 W2 )
2
t R 2 t R1
1.18
W1/ 2 h ,1 W1/ 2 h , 2
W1/2h,1 W1/2h,2 h1/2

W1 W2
66
Resolution Required for
Complete Separation
(tR2 - tR1) (tR2 - tR1)

W1 W2 W1 W2
tR2 - tR1 = W1 = W2 tR2 - tR1 = W1 = W2
RS = 1 RS = 1
If the peaks are isosceles triangles, If the peaks are Gaussian distributions,
they are completely separated. RS > 1.5 is necessary for complete separation.
67
 Relationship Between
Resolution and Other
Parameters
The resolution is a
function of the t R 2 t R1
RS
separation factor, 1
(W1 W2 )
the theoretical plate 2
number, and the
1 1 k2
retention factor. N
The separation can 4 k2 1
be improved by
improving these 3
parameters!
68
 Contribution of Capacity
Factor to Resolution
Increasing the 1.0

Contribution ratio for resolution

capacity factor
0.8
improves separation!
0.6
A capacity factor of
around 3 to 10 is 0.4

appropriate. 0.2
Exceeding this just
0.0
increases the 0 5 10 15 20
analysis time. Capacity factor

69
 Contribution of
Theoretical Plate Number
to Resolution
The resolution
increases in
proportion to
the square root
of the
theoretical plate
number.

70
 To Improve Separation...

Before
adjustment

k increased Eluent replaced with one

of lower elution strength.

Column replaced with one of

N increased superior performance.
Column lengthened.

Column (packing material) replaced.

Eluent composition changed.
increased Column temperature changed.
71
 The factors which influence
the HPLC performance
1. Internal diameter of column
- the smaller in diameter, the higher in
sensitivity
2. Pump pressure
- the higher in pressure, the higher in
separation
3. Sample size
4. The polarity sample, solvent and column
5. Temperature
- the higher in temperature, the higher in
separation
Advantages
1. Needs a small sample with a high
accuracy and precis
2. Non-destructed sample during
operation compared to GC.
Disadvantages
1. Need a skill to run the
instruments
2. Solvents consuming
Comparison of HPLC and GC

74
 The Chromatogram
to - elution time of unretained peak
tR- retention time - determines sample identity
tR

tR
mAU Area or height is proportional
to the quantity of analyte.

to

Injection time
75
1.Retention time:
Retention time is the difference in time between the point of injection and
appearance of peak maxima. It is also defined as time required for 50% of a component to
be eluted from a column. It is measured in minutes and seconds.

2.Retention volume:
Retention volume is the volume of carrier gas required to elute 50% of the
component from the column. It is the product of retention time and flow rate.
Retention volume = Retention time flow rate
3.Seperation factor:
Separation factor is the ratio of partition coefficient of the 2 components
to be separated.

S=Ka/Kb=(tb-to)/(ta-to)

Where to = Retention time of unretained substance

Ka, Kb = Partition coefficients of a, b
ta, tb = Retention time of substance a, b

If there is a more difference in partition coefficient between 2 compounds,

the peaks are far apart and the separation factor is more. If the partition
coefficient of 2 compounds are similar, then the peaks are closer and the
separation factor is less.
4. Resolution:
Resolution is the measure of extent of separation of
2 components and the base line separation achieved.

Rs = 2 (Rt1-Rt2) / w1+w2
5. Height Equivalent to a Theoretical Plate (HETP):
A theoretical plate is an imaginary or hypothetical
unit of a column where distribution of solute between
stationary phase and mobile phase has attained equilibrium.
It can also be called as a functional unit of the column.
 A theoretical plate can be of any height, which describes the
efficiency of separation. If HETP is less, the column is more efficient. If
HETP is more, the column is less efficient.
HETP = length of the column/ no. of theoretical plates
HETP is given by Van deemter equation

HETP = A+B/u+Cu

Where A = Eddy diffusion term or multiple path diffusion which arises due to the packing
of the column. This can be minimized by uniformity of packing.
B = Longitudinal diffusion term or molecular diffusion.
C = Effect of mass transfer.
u = flow rate or velocity of the mobile phase.
 6. Efficiency:
Efficiency of a column is expressed by the theoretical plates.
n = 16 Rt2/ w2

Where n = no of theoretical plates

Rt= retention time
w = peak width at base

Rt and w are measured in common units (cm or mm , min or sec ). No of

theoretical plates
Is high, the column is said to be highly efficient. For GLC, a value of
600/metre is sufficient. But in HPLC, high values like 40,000 to
70,000/ meter are recommended.
 7. Asymmetry factor:
A chromatographic peak should be symmetrical
about its centre and said to follow Gaussian distribution.
But in practice due to some factors, the peak is not
symmetrical and shows tailing or fronting.
Fronting is due to saturation of stationary phase
and can be avoided by using less quantity of sample.
Tailing is due to more active adsorption sites and can be
eliminated by support pretreatment.
Asymmetry factor (0.95 to 1.05) can be calculated
by AF = b/a (b, a calculated by 5% or 10% of the peak
height).
 Modes of High Performance
Liquid Chromatography
Types of Compounds Mode Stationary Mobile Phase
Phase
Neutrals Reversed C18, C8, C4 Water/Organic
Weak Acids Phase cyano, amino Modifiers
Weak Bases

Ionics, Bases, Acids Ion C-18, C-8 Water/Organic

Pair Ion-Pair Reagent

Compounds not Normal Silica, Amino, Organics

soluble in water Phase Cyano, Diol

Ionics Inorganic Ions Ion Anion or Cation Aqueous/Buffer

Exchange Exchange Counter Ion
Resin

High Molecular Weight Size Polystyrene Gel Filtration-

Compounds Exclusion Silica Aqueous
Polymers Gel Permeation-
Organic

82
 HPLC Applications
Bioscience
Chemical proteins
peptides
polystyrenes nucleotides
dyes
phthalates

tetracyclines
Pharmaceuticalscorticosteroids
antidepressants
barbiturates Consumer Products
lipids
antioxidants
sugars
Environmental
polyaromatic hydrocarbons Clinical
Inorganic ions amino acids
herbicides vitamins
homocysteine

83
 Application of HPLC
1. Pharmaceuticals industry
To control the drug stability
Quantity of drug determination from
pharmaceutical dosage forms, ex. Paracetamol
determination in panadol tablet
Quantity of drug determination from biological
fluids, ex: blood glucose level

2. Analysis of natural contamination

- Phenol & Mercury from sea water

3. Forensic test
- Determination of steroid in blood, urine &
sweat.
- Detection of psychotropic drug in plasma
 Application of HPLC
4. Clinical test
- Monitoring of hepatic chirosis patient
through aquaporin 2 in the urine.

5. Food and essence manufacture

- sweetener analysis in the fruit juice
- preservative analysis in sausage.
 Quantitative Analysis
Quantitation performed with peak
area or height.
Calibration curve created beforehand
using a standard.
Absolute calibration curve method
Internal standard method
Standard addition method

86
 Calibration Curve for
Absolute Calibration Curve
Method
Area
Concentration
A1 Calibration curve
C1
A4

A2

Peak area
A3
C2

A2
A3
C3
A1

A4
C4 C1 C2 C3 C4
87 Concentration
 Calibration Curve for
Internal Standard Method
Concentration Area

Area for target substance / Area for internal standard

Target Internal
substance standard A1 AIS Calibration curve
C1 CIS A4 /AIS

A2 AIS A3 /AIS
C2 CIS

A2 /AIS
A3 AIS
C3 CIS
A1/AIS

A4 AIS
C4 CIS C1/CIS C2 /CIS C3 /CIS C4 /CIS
Concentration of target substance /
88
Concentration of internal standard
 Advantages of Internal
Standard Method (1)
Not affected by inconsistencies in injection
volume.
IS
X AX / AIS
10 L
injected

Same area
ratio
IS
X
9 L
injected
CX / CIS
89
 Advantages of Internal
Standard Method (2)
Not affected by the pretreatment
recovery rate.
IS
X

AX / AIS
100%
recovery
rate

Same area
ratio
IS
90% X
recovery
rate
CX / CIS
90
Selection Criteria for Internal Standard

It must have similar chemical properties

to the target substance.
Its peak must appear relatively near that
of the target substance.
It must not already be contained in the
actual samples.
Its peak must be completely separated
from those of other sample components.
It must be chemically stable.

91
 Example:
Substances A and B have retention times of 16.4 and 17.63 min; respectively,
on a 30-cm column. An unretained species passes through the column in 1.30
min. The peak widths (at base) for A and B are 1.11 and 1.21 min, respectively.
Calculate: (a) column resolutIon, (b) average number of plates in the column,
(c) plate height, (d) length of column required to achieve. resolution of 1.5, and
(e) time required to elute substance B on the longer column.

(a.) Calculate the column resolution.

Rs = (2((tR)y - (tR)x))/(Wx + Wy)
Rs = (2(17.63 min - 16.4 min))/( 1.11 min + 1.21 min) = 1.06

(b.) Average number of plates N = 16 * (tR/W)2

for peak A N = 16 * (16.4/1.11)2 = 3493 plates
for peak B N = 16 * (17.63/1.21)2 = 3397 plates
Average av N = (3493 + 3397)/2 plates = 3445 plate
(c.) Height equivalent of a theoretical plate
H = L/N = (30 cm/3445 plates) = 0.0087 cm/plate
 References
http://192.215.107.101/ebn/942/tech/techfocus/1071main.html
http://www.chem.usu.edu/~sbialk/Classes/565/opamps/opamps.
html
Skoog, Holler, and Neiman. Principles of Instrumental Analysis.
5th ed. Orlando: Harcourt Brace & Co., 1998.
http://weather.nmsu.edu
http://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/hplc.htm
http://www.chemistry.nmsu.edu/Instrumentation/Lqd_Chroma.ht
ml
http://weather.nmsu.edu/Teaching_Material/SOIL698/Student_M
aterial/HPLCHP1090/HPLCINJ.HTM
http://test-
equipment.globalspec.com/LearnMore/Labware_Scientific_Instru
ments/Analytical_Instruments/Chromatographs/HPLC_Columns
http://www.chemistry.adelaide.edu.au/external/soc-
rel/content/lc-col.htm