High Performance Liquid

Chromatography
Kromatografi Cair Kinerja Tinggi

1
 HPLC
C
P I

FD

1. Fase gerak dipompa masuk ke tabung

stainless steel yg berisi partikel fase
E diam berdiameter 3-10 m
L
2. Dengan injektor Rheodyne larutan
baku/sampel disuntikan ke dalam
aliran fase gerak
3. Pemisah komponen terjadi ditabung fase diam
4. Komponen dideteksi oleh detektor
5. Data diproses oleh komputer
2
 Injektor Rheodyne
dari pompa

ke kolom putar tuas

SUNTIK

dari pompa ke kolom

A B

F C

E D A B

F C
E D

ISI LOOP
dgn semprit
suntik
 Penggunaan

Gabungan HPLC dengan detektor spektrofotometer dengan DAD

menjadikan HPLC sebagai
• metoda baku analisis sediaan farmasi yang akurat, persis dan tegar,
bagi industri farmasi.
• metoda untuk memantau kestabilan atau mengetahui terjadinya
degrasi bahan obat murni, maupun kestabilan bahan aktif dalam
sediaan farmasi
• metoda untuk mengukur kadar bahan aktif dan metabolitnya di dalam
cairan biologis
• Metoda penetapan nilai koefisien partisi dan pKa dari suatu obat dan
penetapan obat yang terikat dengan protein dalam cairan biologis

4
 Strengths

• Easily controlled and precise sample introduction ensures quantitative

precision.
• HPLC is the chromatographic technique which has seen the most
intensive development in recent years leading to improved, columns,
detectors and software control
• The variety of columns and detectors means that the selectivity of the
method can be readily adjusted.
• Compared to GC there is less risk of sample degradation because
heating is not required in the chromatographic process
• Readily automated.

5
 Limitations

• There is still a requirement for reliable and

inexpensive detectors which can monitor
compounds that lack a chromophore
• Drugs have to be extracted from their
formulations prior to analysis
• Large amounts of organic solvent waste is
generated, which is expensive to dispose of

6
 Modes of HPLC
• Normal Phase mode
• Reverse Phase mode
• Reverse Phase Ion Pairing mode
• Ion Exchange mode
• SEC mode ( GPC / GFC )
• Chiral separation mode

7
 Reverse Phase HPLC Columns
• C18 (ODS) type
• C8 (octyl) type
• C4 (butyl) type Non-polar property
• Phenyl type -Si-C18H35
• TMS type
• Cyano type Si

8
 Structural Factors Which Govern Rate of Elution 0f
Compounds from HPLC Columns
• Elution of neutral compounds
– For a neutral compound it is the balance between its polarity and Iipophilicity
which will determine the time it takes for it to elute from an HPLC column; the
pH of the mobile phase does not play apart. In the case of a reverse-phase
column, the more lipophilic a compound is the more it will be retained
►
• Ionizable compounds

9
 Hydrophobicity

• If the sample has

– CH3CH2CH2--- : Carbon chain Hydrophobicity
– : Aromatic group becomes strong.

• If the sample has

-COOH : Carboxyl group
-NH2 : Amino group Hydrophobicity
-OH : Hydroxyl becomes weak.
group
10
Retention Time and Hydrophobicity

OH

C18 (ODS)
Weak

Strong
OH

11
 Increase of solvent polarity
20 % 30 % 40% / H2O

1 : p-Hydoxymethylbenzoate
2 : p-Hydoxyethylbenzoate Solvent : MeOH
3 : p-Hydoxypropylbenzoate
4 : p-Hydoxybutylbenzoate 12
Effect of stationary phase

C8

Medium
C18 (ODS)
sample
Strong C4
sample
Weak
sample

13
 Effect of stationary phase

 Analytical Conditions
ODS C8 TMS  Column : Shim-pack CLC-ODS
 Mobile phase : MeOH : H2O = 7 :3
 Flow rate : 1.0 mL/min
 Temperature : 40 C
 Injection volume : 10 uL
 Detection : UV-254 nm
 Peaks
1. Methyl benzoate
2. Ethyl benzoate
3. n-Propyl benzoate
4. n- Butyl benzoate

14
• Ionizable compounds
– Control of elution rate of ionisable compounds by adjustment of pH of mobile
phase. Untuk asam K’app = K’/(1+10pH-pKa) untuk basa K’app = K’/(1+10pKa-pH) ►
– Mobile-phase conditions may be selected in straight-phase chromatography
where the ionisation of the analytes is suppressed, and basic compounds are run
in a basic mobile phase and acidic compounds are run with an acidic mobile
phase
– The pH of the mobile phase can only be set within the range of ca 2-8.5 pH
units because of the tendency for extremes of pH to dissolve silica gel and
break the bonds between silane-coating agents and the silica gel support

R-COOH  RCOO- + H+
(pKa=4.5)
R-NH2 + H+  R-NH3+
(pKa=6.0)

15
 Reverse Phase
Ion-Pair Chromatography

Ion-Pair Reagent

16
 Ion Paring
Important Considerations
• Type of Ion-Pair reagents
• Concentration of Ion-Pair reagents
• pH of solvent

R-COOH RCOO- + H+
(pKa=4.5)
R-NH2 + H+ R-NH3+
(pKa=6.0)
17
 Type of Ion-Pair Reagents

Hexane Sulfonate Pentane Sulfonate

Mobile Phase: H2O/MeOH

1:1,with 0.005M ion pairing
reagent and 1% HOAc

1 Maleic Acid
2 Phenylephrine
3 Phenylpropanolamine
4 Naphazoline
5 Phenacetin
6 Pyrilamine

18
 Concentration of
Ion-Pair Reagents

19
 Causes of Tailing Peaks
• Built-up of garbage on the column inlet
• Extra column effects (dead volume)
• Sample Overload
• Incorrect solvents for the sample
• Secondary retention effects
– Silanol group
– Residual heavy metal

20
 Dead Volume

• Dead volume will cause a broad peak.

– Sample Injector Connection
– Column Connection
– Detector Connection
Good

Dead Volume
21
 Incorrect solvent for sample
• Better do not select a high soluble solvent as a sample
solvent.
Methanol as a sample solvent Ethanol as a sample solvent

20 uL Caffeine 20 uL Caffeine

Ethanol as a sample solvent

Better inject small
10 uL Caffeine amount of sample.

22
 Secondary retention effects

• Silanol Group
– Even modified silica gel (e.g. ODS, C8), residual
silanol group are still remained on the surface
area.
– Silanol group will strongly absorb amine
compounds, therefore tailing will be happened.
C18
OH
Silanol group
silica core C18
O- negative charge
C18 23
C18
 End capping

• To suppress the silanol group effect, an end

capping type of column will be selected.

C18 C18
OH TMS treatment O-TMS
silica core C18 silica core C18
OH TMS : trimethylsilyl group
O-TMS
C18 C18
C18 C18
[Non-End capping type] [End capping type]
24
 Secondary retention effects

• Residual heavy metal

– Normal silica gel has heavy metal as an impurity.
This heavy metal will have an interaction with
chelate compounds, therefore tailing will be
happened.

C18
M+ O-TMS High pure type of
silica core C18 O
+ O-TMS silica gel are available.
M C18 O
C18
25
 HPLC system

• Isocratic elution system

– Single solvent of constant composition
• Gradient elution system
– Multi solvents of changeable composition
• High pressure gradient system
• Low pressure gradient system

26
Isocratic Elution System

pump injector oven

detector

column

Single Solvent
27
Gradient Elution System

A
column
injector detector
pump oven

B concentration
B

pump

Time
28
 Isocratic Elution Mode

MeOH / H2O = 6 / 4
Long Time Analysis

Bad Separation

MeOH / H2O = 8 / 2

( column : ODS type ) 29

 Gradient Elution Mode

MeOH concentration
95%

30%

30
 Calibration method

• External calibration method

• Internal calibration method
• Standard additive method
• Correction Normalization method

31
 External Standard Calibration
Preparation of Standards

Target Compounds

Dilution Dilution Dilution Dilution

32
 External Standard Calibration
Calculation of Results

Y = bX + a
[Concentration]

b : SLOPE
125 ppm a : Y intercept

2500
2500
[Peak Area]

33
 Internal Standard Calibration
Preparation of Standards

Internal
Target Compounds Standard

Dilution Dilution Dilution Dilution

34
Internal Standard Calibration
Analysis of Vanillin

35
 Internal Standard Calibration
Calculation of Results
[Target Area / IS Area]

Y = bX + a
b : SLOP
5.0 a : Y intercept

1.67 T 2500
500
[Target Conc. / IS Conc.] IS
Y = Target Conc. / IS Conc.
1.67 = Target Conc./ 100 ppm
Target Conc. = 167 ppm
36
 Data kalibrasi standar internal
Kadar Luas Puncak Rasio
Is St Is St
ppm ppm Count Count Kadar Area
10.02 14.20 627 662
10.02 17.20 628 809
10.02 20.04 627 939
10.02 23.1 609 1463

10.02 609 900

10.02 616 895
10.02 618 905

37
 Data Kalibrasi Internal
Kadar Area Puncak Rasio
Is St Is St KadarSt/Is AreaSt/Is
ppm ppm Count Count
10.02 14.2 627 662 1.4172 1.0558
10.02 17.2 628 809 1.7166 1.2882
10.02 20.04 627 939 2.0000 1.4976
10.02 23.1 609 1463 2.3054 2.4023
Sample
10.02 18.06 609 900 1.8023 1.4778
10.02 17.89 616 895 1.7851 1.4529
10.02 17.97 618 905 1.7930 1.4644

r 0.9344
slope (b) 1.4464
intercept (a) -1.1290
38
 Advantage of external standard
calibration method
• Only the target compound separation can be
focused.

Target Target

39
 Disadvantage of external standard
calibration method
• Injection error will directly influence the
quantitative result.
10 uL injection 11 uL injection

100 ppm 110 ppm

40
 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

41
 Advantage of internal standard
calibration method

• Recovery in the pretreatment process can be

estimated. addition of IS (100 ppm)
standard IS (100 ppm) to actual sample
IS T
IS T
1000 950

Recovery = (950/1000)x100 = 95%

42
 Disadvantage of internal standard
calibration method
• Separation is slightly difficult.

IS T IS T

T
IS

43
 Disadvantage of internal standard
calibration method

• It is difficult to look for 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.

44
 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

45
 Standard additive
calibration method

Original
Sample T
Target

T
46
 Standard additive
 ABC   EDC
calibration method AB : BC = ED : DC
100/10 = ED/7
10 = ED/7
ED = 70

T x104
B A
Peak Area
17
T 12

7
C
X =70 ppm
T
E D
-70 0 50 100 ppm
Added amount 47