High Performance Liquid

Chromatography
HPLC

Nia Kristiningrum, M.Farm., Apt

High Performance Liquid
Chromatography

1. Introduction
2. Instrumentation
3. Application
 High Performance Liquid
Chromatography
HPLC is characterized
by the use of high
pressure to push a
mobile phase solution
through a column of
stationary phase
allowing separation of
complex mixtures with
high resolution.
 Keuntungan
 Dapat dilakukan pada suhu kamar
 Kolom dan Detektor bervariasi
 Ketepatan dan ketelitian relatif tinggi
 Mudah dioperasikan secara otomatis
 Analisis kuantitatif yang cepat dengan
presisi dan akurasi yang tinggi
 Dapat diaplikasikan untuk berbagai analit
dalam jenis sampel yang lebih luas
 Kelemahan

 Sulit ditemukan detektor yang universal

 Efisiensi pemisahan lebih rendah daripada
GC
 Lebih rumit dalam pelaksanaannya
Instrument Schematics
Instrument Schematics
High Pressure Liquid Chromatography (HPLC)
Injector
 Macam injector
 Injector dg diafragma (Septum injector)
 Injector tanpa diafragma (Septumless
injection system)
 Injector dengan pipa dosis (loop valve)
 Sistem injeksi otomatis (autoinjector)
 loop valve
 Merupakan pilihan tepat terutama utk
analisis kuantitatif
Auto injector
STATIONARY PHASES
 HPLC Column Categories
 Column Hardware: Standard or Cartridge,
stainless, titanium
 Chromatographic Modes: Normal-phase
(NPC), reversed-phase (RPC), ion-exchange
(IEC), size exclusion (SEC)
 Dimensions : Prep, semi-prep, analytical,
fast LC, micro, nano
 Support Types: Silica, polymer, zirconia,
hybrid
 Column Dimension

Length x ID
Art.-Nr. xxxx HPLC-column 250x3 mm
Lichrospher 100 RP-18 5µm
Particle size in µm

Modification of silicagel

Pore size (Angstrom)

Producer of silica gel

 HPLC Columns
 Column Dimensions
 Length and internal diameter of packing bed
 Short (30-50mm) - short run times, low backpressure
 Long (250-300mm) - higher resolution, long run times
 Narrow ( 2.1mm) - higher detector sensitivity
 Wide (10-22mm) - high sample loading

 Particle Shape
 Spherical or irregular
 Spherical particles  reduced back
pressures and longer column life
 HPLC Columns
 Particle Size
 The average particle diameter,
typically 3-20µm
 Smaller particles offer higher efficiency
but also cause higher backpressure
< 2µm  UPLC

 Surface Area
 Sum of particle outer surface and interior pore
surface, in m2/gram
 High surface area generally provides greater
retention, capacity and resolution
 Low surface area packings generally equilibrate
quickly, especially important in gradient analyses.
 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”
 Normal vs. Reversed Phase

Normal Reverse
Stationary phase polar Non polar
polarity
Solvent polarity Non polar Polar
Elution order Non-polar first, then Polar first, then non-
polar polar
Effect of increasing Increases retention Decreases retention
solvent polarity time time
 Mobile Phases
 Purity
 Detector compatibility
 Solubility of the sample
 Low viscosity
 Reasonable price
 Degassing
 Fase Gerak

 Zatcair yang
digunakan sebagai
fase gerak harus
saling campur.
 Degasser

Menghilangkan udara (gas nitrogen dan oksigen) yang

terlarut.
Adanya udara dapat menimbulkan variasi tekanan pada
pompa atau timbulnys noise pada detektor tertentu
Cara: vacuum degassing, pengaliran gas helium atau
ultrasonikasi.
 Pompa HPLC
 Pompa Pencampur  untuk menarik
solvent fase gerak dari botol reservoir
 Pompa Analisis  mengalirkan fase gerak
ke dalam kolom

 SistemPompa:
 Tekanan Tinggi
 Tekanan rendah
 Pada : HPLC Mengapa perlu tekanan
tinggi?
 Pada HPLC digunakan ukuran partikel packing kolom
(silika) yang sangat kecil, umumnya 3-10um.
 Ukuran partikel makin kecil  luas permukaan makin
besar  transfer massa dari fase gerak ke fase diam atau
sebaliknya makin besar  kolom semakin efisien dan
resolusi peak semakin baik.
 Penggunaan ukuran partikel kecil  diperlukan tekanan
tinggi
 Pada kecepatan alir 0.5 sampai 5 ml/menit, panjang kolom
10-30 cm dibutuhkan tekanan 70-400 atm (1000 – 6000
psi)
Van Deemter Plot (smaller particles have smaller A & C terms)
elution method
1. Isocratic elution
method
2. Gradient elution
method
 Kelemahan sistem elusi isokratik

 Pada awal elusi menghasilkan resolusi yg

kurang baik
 Pada elusi yg lebih lanjut menghasilkan
penambahan lebar puncak dg penurunan
tinggi puncak
 Membutuhkan waktu analisis yg lama
 Kelebihan sistem elusi gradien

 Memungkinkan untuk menghasilkan

kromatogram yg ideal
 Menghasilkan resolusi yg optimum antar
puncak
 Menghasilkan lebar puncak yg seragam
untuk semua puncak
 Waktu analisis lebih pendek
Gradient elution method : komposisi
eluen berubah terhadap waktu

For separation of a sample containing many components

MeOH(100)

Gradient
Mobile phase

MeOH/Water 0.5M
(50/50)

0.1M KH2PO4

Step wise
0.01M

0 5 10 15 20 25
Time(min)
Gradient elution method : memperbaiki
resolusi dan mempercepat analisis
Advantage of gradient elution method

Isocratic elution method Gradient elution method

A
A
Finepak SIL C18
MeOH/1% AcOH(40/60)
B

B
*
*
MeOH/1% AcOH
A
MeOH/1% AcOH(30/70) 30/70→45/55
Linear Gradient、16min

A : Chlorogenic acid
B : Rutin
B
* : Impurity
 High Throughput Gradient Analysis on Rapid
Resolution with Low and High Flow Rates
Columns: ZORBAX Rapid Resolution HT SB-C18, 2.1 x 30 mm, 1.8 mm
Mobile Phase: A: 20 mM Na2HPO4 pH 2.8, B: ACN
Gradient: A: 30 – 70%B in 5 minutes, hold for 1min. B: 30 – 80% B in 1.1 min, hold for 0.3 min, Flow Rate: see below,
Temperature: Ambient Autosampler: 1100, Bypass Mode Detection: UV 230 nm
Sample: 1. Estriol 2. Estradiol 3. Ethynylestradiol 4. Dienestrol 5. Norethindrone

A. 0.25 mL/min

0 1 2 3 4 min
5
B. 0.75 mL/min

0 0.2 0.4 0.6 0.8 1 min

1.2

• High flow rates reduce analysis time and increase throughput in gradient
separations as well as isocratic separations.
4. Gradient elution method

Precautions in gradient elution method

- Can the gradient save time ?

- Reproducibility
- Baseline
- Ghost peak
 Solvent Extraction (pH effects)
 Equilibrium constant for this partitioning is
K (partition coefficient)

[S]2
K=
[S]1
 Solvent Extraction (pH effects)
 with organic acids/bases:

Ka
HA H+ + A-
Kb
B + H2O BH+ + OH-
Generally, neutral species are more soluble
in an organic solvent and charged species
are more soluble in aqueous solution
 Solvent Extraction (pH effects)
 Partitioning of organic acids between two
phases:
very little here, ions
have poor solubility
organic HA H+ + A-

Ka
aqueous HA H+ + A-
 Solvent Extraction (pH effects)
 When the solute (acid/base) can exist in different
forms, D (distribution coefficient) is used instead
of K (partition coefficient)
 Solvent Extraction (pH effects)

total conc. in phase 2

D=
total conc. in phase 1
HA [HA]org
D=
K [HA]aq + [A-]aq
Ka
HA H+ + A-
 Solvent Extraction (pH effects)
 pH effect on D for organic acids
[H+]=Ka
[H+]>>Ka
pH=pKa
K

mainly mainly
D HA A-

[H+]<<Ka
[HA]org
D=
[HA]aq + [A-]aq pH
 Solvent Extraction (pH effects)
 Analogous treatment for organic bases

[H+]=Ka
pH=pKa [H+]<<Ka
K
K Ka mainly mainly
D= D BH+ B
[H+] + Ka
[H+]>>Ka

pH
 Solvent Extraction (pH effects)
 Example problem: Want to separate two organic
acids using a scheme based on pH.
Acid 1 (pKa = 4), Acid 2 (pKa = 8)

K1 Acid 2 stays in
K2 organic phase,
acid 1 is extracted
D into aqueous phase

4 pH 8
 HPLC System
9060 Polychrom Computer
(Diode Array) Detector Workstation

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

Rheodyne
Injector

Keep an eye on
HPLC these 4 screens!
Column
Solvent Delivery System
 %A %B %C Flow Rate Pressure to column
{H2O} {MeOH} (mL/min) (atmos.)
load

Ready
inject

Rheodyne
Injector
Varian 9010 Solvent Delivery System to injector

through pump

Column
through C
pulse
dampener
A B

from solvent to
Ternary Pump reservoir detector
Variable UV/Vis Detector
ABS AUFS l RunTime EndTime
0.001 2.000 238 0.00 min 10.0 min

Ready
 Ready
UV Spectrum
UV Spectrum {shows full UV abs.}
UVmax
UVmax

Chromatogram

ABS.
Reset
Wavelength

Rt Rt

ABS.
Time

Chromatogram
{shows peaks, Rt}

Varian 9060
Polychrom Detector
 Approximation
HPLC Chromatograms of peak area by
triangulation
Absorbance 

Peak A Peak B

height

0 1 2 3 4 5 6 7
Time (minutes) base

Rt = 3.0 min. Rt = 5.2 min. base x height

faster moving slower moving Area =
2
less retained more retained
Detector
 Syarat Detector
 Sensitivitas tinggi
 Kestabilan dan reprodusibilitas baik
 Memberi tanggapan linier thd konsentrasi analit
 Dpt bekerja pd suhu kamar ad 400oC
 Tidak dipengaruhi oleh prubahan suhu
 Mudah digunakan
 Selektif
 Tdk merusak sampel
5. HPLC detectors
HPLC detectors
UV-VIS(Absorption) / PMT UV-VIS

PDA (Absorption)

Differential refractometer(Refractive index)

Fluorometric (Florescence)

Electrochemical (ECD) (Oxidation -reduction)

Conductivity

Mass

Chiral (OR)
5. HPLC detectors
UV/Vis detector
- Selective detection minimizing effects from other components

-High sensitivity detection at maximum absorption wavelength

 PHOTOMULTIPLIER TUBE
The photomultiplier tube is a commonly used
detector in UV-Vis spectroscopy. It consists of a
photoemissive cathode (a cathode which emits
electrons when struck by photons of radiation),
several dynodes (which emit several electrons for
each electron striking them) and an anode.
 PHOTOMULTIPLIER TUBE
The Variable Wavelength UV Detector uses a monochromator (slits and a
grating) to select one wavelength of light to pass through the sample cell.
 PHOTODIODE ARRAY
The linear photodiode array is an example of a multichannel photon
detector. These detectors are capable of measuring all elements of a beam
of dispersed radiation simultaneously.

A linear photodiode array comprises many small silicon photodiodes

formed on a single silicon chip. There can be between 64 to 4096 sensor
elements on a chip, the most common being 1024 photodiodes. For each
diode, there is also a storage capacitor and a switch. The individual diode-
capacitor circuits can be sequentially scanned.

In use, the photodiode array is positioned at the focal plane of the

monochromator (after the dispersing element) such that the spectrum falls
on the diode array. They are useful for recording UV-Vis. absorption
spectra of samples that are rapidly passing through a sample flow cell,
such as in an HPLC detector.
 PHOTODIODE ARRAY
The Photodiode Array Detector passes all wavelengths of light through the
sample cell, then focuses each wavelength on a single sensor element.
5. HPLC detectors
Multi-wavelength detector (PDA detector)
3D chromatogram
5. HPLC detectors
Multi-wavelength detector
Cont. data
5. HPLC detectors
Features of Multi-wavelength detector

1. Spectrum collection at any time

2. Library search
3. Purity check
4. Quantitative analysis at 6 wavelengths
5. HPLC detectors
Improved sensitivity
Saccharin (SAC) and sorbin acid (SOR)

230nm 265nm

SOR

SOR
0nm
SAC

SAC
12.250

20－
10－

20－

10－

12.467
3.575

Wavelength programming 3.608

Fixed wavelength at 265nm
5. HPLC detectors
UV spectrum measurement
to find wavelength effective for wavelength programming
1.0

Diazepam
(DZP)

Absorbance
Nitrazepam
(NZP)
0.5

Chronazepam
(CZP)

210 250 300 350 0

Wavelength(nm)
5. HPLC detectors
Wavelength programming and fixed wavelength

Blood serum
NZP : 420ng/ml
CZP : 130ng/ml
DZP : 440ng/ml

310nm 250nm

NZP
DZP
NZP

DZP
CZP
CZP

0 5 10 0 5 10

Wavelength programming Fixed wavelength

 Peak Purity test: HPLC

 Spektra UV/Vis analit dan zat standar

(authentic reference material) dengan diode
array detector  overlay 
Evaluasi korelasinya (r, MF, FTIR, MS)

 Pengukuran spektra pada “upslope,

apex dan down slope”
 Peak harus “pure”
 Match Factor
 MF = 1000  r = 1 100% pure peak
 MF > 990  pure
 MF < 900  not pure
 900 < MF < 950  contaminated
 Pure and Impure HPLC peaks

• Peak purity tests can also be evaluated with

– The 3D-spectra of Photodiode array detectors
– Mass spectrometry
5. HPLC detectors
Principle of Fluorescence detector

(S1)

Excited state(S2)

(S3)

excitation emission Hν (fluorescence)

Ground state（S0）

Mobile phase
5. HPLC detectors
Features of Fluorescence detector

1. Selective detection
2. Detection at any excitation or emission wavelength
3. High sensitivity
5. HPLC detectors
Wavelength programming by Fluorescence detector
Wavelength programming
0 6.6 10.0 min
Ex 275 240 450
Em 400 350 525 nm Fixed wavelength Fixed wavelength
Ex=275nm Ex=450nm
Em=400nm Em=525nm

VB2
VB2 Phosphate

VB2 Phosphate
VB6

VB2
VB6
VB1

0 10 20 min 0 10 20 min 0 10 20 min

Column : Finepak SIL C18S

Mobil phase : MeOH/Phosphate Buffer Gradient
5. HPLC detectors
Selectivity of
UV detector and Fluorescence detector

UV detector

Fluorescence detector
5. HPLC detectors
Principle of RI detection

light light
i i

r0 r

Solvent Sample and solvent

r0>r
this detector can be very useful for detecting those compounds
that are nonionic, do not adsorb in the UV, and do not fluoresce
Refractive Index Detector
5. HPLC detectors
Considerations for RI detection

1. Temperature change
2. Pressure
3. Flow rate
5. HPLC detectors
Detectors

UV Fluorescence RI

Sensitivity ng pg μg

Detection selective highly selective universal

selectivity

Temperature small small large

Influence

Gradient elution possible possible impossible

 GANGGUAN ANALISIS
 Gangguan sistem garis dasar (baseline)
 Gangguan bentuk puncak (peak shape)
 Gangguan baseline
 Noise (derau)
 Drift (melayang)
 Spiking (berpaku)
 Wander (menyimpang)
 Shift (bergeser)
 Gangguan peak shape
 Puncak lebih kecil dr yg diharapkan
 Tailing
 Fronting (puncak mengandung)
 Puncak membelah (split)
 Puncak negatif
 Puncak berubah bentuk (cigar top, round
top, flat top)
Prosedur Analisis

Laboratory

Quality Assurance:
ISO 17025:2005
ISO 9001:2008

Analisis di Laboratorium

Hasil Analisis
Sampel
Ekstraksi, Pemurnian (Clean-up); analisis
dengan instrumen
 Preparasi Sampel
 Larutan  Siap disuntikkan, diencerkan atau ditambah
dapar/standar internal lebih dahulu
 Padat  Perlu dilarutkan terlebih dahulu
Saring (0.2 – 0.45 μm) sebelum disuntikkan

Solid Handling  grinding, milling, homogenization

Extraction  Shaking, ultrasonication, Soxhlet, Liquid-liquid Extraction,
Solid Phase Extraction (SPE)
Liquid Handling  Pipetting, dilution, concentration, pH/ionic strength
adjustment
Phase separation  filtration, centrifugation, precipitation
Sample clean-up: Column chromatography, SPE
Derivatization: pre or post column derivatization
 Contoh Kondisi HPLC
 Fase gerak: ?
(elusi isokratik/gradien?)
 Jenis kolom: ?
 Kecepatan alir:?
 Detektor:
(UV, Panjang gelombang ?)
 Analisis Kualitatif HPLC
 Berdasarkan waktu retensi
 tR A = tR B  zat A mungkin sama dengan B
 tR A tidak sama dengan tR B  zat A tidak sama
dengan zat B
 Untuk sampel kompleks tR dipengaruhi banyak
faktor  gunakan waktu retensi relatif (RRT):
 tR analit dibandingkan tR komponen pembanding
dalam sampel
 Penambahan zat standar ke dalam sampel
 Data spektra dari PDA (match factor) atau MS
 Quantitative Analysis

 Normalization method
 External standard method
 Internal standard method
 Standard addition method
Quantitation by Normalization Method

All the analytes present in the sample must elutes from the
column, with enough resolution and, furthermore have to be
detected by the available detector.

The peak area is proportional to the weight of the analytes

having passed through detector cell

The analytical signals lies within the linearity ranges

Thus,
Percentage in weight of each analyte
% i = Ai /  Ai X 100
Quantitation by External Standard

This method is the most general method for determining the

concentration of an analyte in samples
It involves the construction of a calibration plot (Area or
height vs. Analyte concentrations) by using some
concentrations (minimum 4 concentrations) of external
standard; the concentration of the analyte can be
determined by “interpolation”. It is not recommended to do
“extrapolation”.
 External Standard Calibration
Calculation of Results

Y = bX + a
b : SLOPE
a : Y intercept
[Concentration]

125 ppm

2500
2500

[Peak Area]
 Disadvantage of external
standard calibration method
 Injection error will directly influence the
quantitative result.

10 uL injection 11 uL injection

100 ppm 110 ppm

 Advantage of internal standard
calibration method
 Injection error can be eliminated.

10 uL injection 11 uL injection
2200
2000 1100
1000 T
IS
IS T

2000 / 1000 = 2 2200 / 1100 = 2

 Disadvantage of internal
standard calibration method
 Separation is slightly difficult.

IS
T IS T

T
IS
 Disadvantage of internal
standard calibration method
 It is difficult to find the IS compound.
 The chemical structure of IS compound is similar
with one of target compound.
 IS sample is not existent in the actual sample.
 Calibration Method

 External standard calibration

 Separation is not difficult
 Injection error will directly influence the
quantitative result
 Internal standard calibration
 Injection error can be eliminated
 Recovery in the pretreatment procedure can be
estimated
 Separation is slightly difficult
 Difficult to look for the IS compound
 Contoh Soal HPLC.1:
Kondisi Analisis HPLC
 Fase Diam : Kolom RP C18
 Fase Gerak : Metanol : air pH 3,2 =
20 : 80
 Volume Injeksi : 2ml
 Panj Gel : 260 nm
 Flow rate : 1 ml/menit
Pada kondisi analisis tersebut didapat kromatogram
sbb:
 Rt  A=2,0 B=4,0 C=6,0
 w  A=0,3 B=0,8 C=0,4
 Contoh Soal HPLC.1:
Kromatogram standar
Kromatogram blangko

A
B

2 4 6 8 menit 2 4 6 8 menit

Kromatogram sampel

Pertanyaan:
A
B
1. Apakah Kondisi analisis tsb selektif
? jelaskan!
C 2. Hitung Resolusi A thd B dan B thd
C pada kromatogram sampel!

2 4 6 menit
8 min
 Contoh Soal HPLC.2:
 Komposisi : tiap 5 ml sirup mengandung :
amoksisilin trihidrat setara dengan amoksisilin 250,0 mg
Kalium klavulanat setara dengan asam klavulanat 62,5 mg
 Persyaratan USP 25 : Amox and Clavulanate Potassium for oral
suspension contains the equivalent of not less than 90,0% and not more than
120,0% of the labeled amount of amoxicillin and the equivalent of not less
than 90,0% and not more than 125,0% of the labeled amount of clavulanic
acid
 Martindale 33th Ed
 1,15 gram of Amoxicillin trihydrate is approximately equivalent to 1 g of
amoxicillin
 1,19 gram of Potassium Clavulanate is approximately equivalent to 1 g of
clavulanic acid
 Preparasi sample : ditimbang 360,0 mg sirup (BJ=1.45g/ml) dilarutkan dg
air ad 25,0 ml, divorteks 10 menit, disaring.
 Contoh Soal HPLC.2:
 Data hasil pengamatan ( vol injeksi sample sama dengan volume injeksi
standar) sbb :
Konsentrasi
Luas Area
(ppm)
Identitas Penimbangan
Amox Amox
K Klav K Klav
trihidrat trihidrat
Standar 1 400 100 26587 6568
Standar 2 500 125 33245 8218
Standar 3 600 150 39812 9822
Standar 4 700 175 46527 11500
Sampel
360,5 mg 38589 9891
Replikasi 1
Sampel
360,1 mg 38410 9880
Replikasi 2
Hitung kadar bahan aktif dalam sample sirup % akurasi ?
Apakah kadar sample memenuhi syarat farmakope?
Any Questions,
comments or
suggestions ?