EXPERIMENT 2: HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) METHOD DEVELOPMENT
OBJECTIVE
I.
To identi identify fy the the compon components ents of of the mixt mixture ure usin using g the selec selected ted HPLC HPLC condit condition ions. s.
ABSTRACT
The chromatography process begins by injecting a sample solution into the mobile phase through the injector port. As a sample solution in the mobile phase flows through a column the compounds of the solution will migrate according to the interactions of the components with the stationary phase. The chemical interactions of the mobile phase and sample solution as well as with the stationary phase determine the degree of migration and separation of components contained in the sample. !or example the components that ha"e stronger interactions with the mobile phase than with the stationary phase will elute faster from the column and ha"e shorter retention time compared to those components which ha"e stronger interactions with the stationary phase. The type and composition of the mobile phase is one of the influence in separation of the components. Therefore the mobile phase can be altered in order to manipulate the interactions of the sample components and the stationary phase. In isocratic elution components c omponents are eluted using constant mobile phase composition. In grad gradien ientt eluti elution on diff differe erent nt comp compon onen ents ts are are elut eluted ed by chan changi ging ng the the mobi mobile le phase phase composition as the separations proceeds. #y increasing the strength of the mobile phase will subse$uently results in elution of retained components.
INTRODUCTION
High performance li$uid chromatography is the most widely used of all of the analytical separation techni$ue. It%s suitable for separating non"olatile species or thermally fragile ones. Part Partit itio ion n chro chroma mato togr grap aphy hy is the the most most wide widely ly used used of all all the the four four type typess of li$u li$uid id chromatogra chromatography phy procedure. procedure. It di"ides di"ides into two& normal'phase normal'phase chromatography chromatography and re"erse' re"erse' phase chromatography. chromatography. !or this analysis we used re"ersed phase chromatography. In re"erse'phase chromatography the stationary phase is non polar and the mobile phase is relati"ely polar. The most polar compon component ent will elute elute first first and increasing the mobile phase polarity increase the elution time. (ethod de"elopment tends to be more complex in li$uid chromatography because the
sample components interact with both the stationary phase and the mobile phase. )uccessful chromatography with interacti"e mobile phase re$uires a proper balance of intermolecular forces among the three acti"e participants in the separation process' the solute the mobile phase and the stationary phase. These intermolecular forces are described $ualitati"ely in term of the relati"e polarity of three reactants. The polarities of "arious analytes functional groups in increasing order are* hydrocarbon +ether +ester + ,etones + aldehyde + amides + amines + alcohols. -ater is more polar compounds than compounds containing any of the preceding functional groups. ften in choosing a column for a partition chromatographic separation the polarity of the stationary phase is matched roughly with that of the analytes& a mobile phase of considerably different polarity is then used for elution. This procedure is generally more successful than one in which the polarities of the solute and mobile phase are matched but different from that of the stationary phase. Here the stationary phase often cannot compete successfully for the sample components& retention time becomes too short for practical application. At the other extreme of course is the situation where the polarity of the solute and stationary phase are too much ali,e and totally different from that of the mobile phase. Here the retention times becomes inordinately long. In summary polarities for solute mobile phase and stationary phase must be carefully blended if good partition chromatography separation are to be reali/ed in a reasonable time. 0nfortunately theories of mobile phase and stationary phase interaction with any gi"en set of sample component are impacted and at best we can only narrow the choice of stationary phase to a general type. Ha"ing made this choice we then perform a series of set trial and error experiment in which chromatogram are obtained with "arious mobile phase until a satisfactory separation is reali/ed. If resolution of the entire component of a mixture pro"es to be impossible different types of column may ha"e to be chosen.
ANALYTICAL PROCEDURE
a. Instrument set up 1may "ary depending on instrument2* 3etector wa"elength * 456 nm !low rate * 7.5 mL8min (obile phase * acetonitrile * water b. 9ffect of mobile phase on HPLC separation i. The instrument is set to use a mobile phase ratio of acetonitrile*water 15:*5:2 ii.
and the sample is injected. Then the mobile phase is changed the mobile phase composition to ;:*<:. The suitable composition of mobile phase for the separation of the compound is determined.
c. Identification of components in the mixture 9ach compound is injected indi"idually and the components of the mixture is identified by using HPLC conditions. d. )eparation using gradient elution The gradient elution is performed based on the separation to impro"e the efficiency of the column.
RESULT
!= I)C=ATIC 9L0TI>& =esponse !actor
Area
(obile Phase =atio
=etention Time 1minute2
;?.<;@4 ;?<;.@45 4B.7@5:
[email protected]:7 4@.;@;5 4@;@.;57 45.<<6< 45<<.6<: @;B.6?4 @;B6?.4: )ample I3* )tandard mixture 17:: ppm2 =espond !actor 1=!2
5: H4 * 5: AC>
7.74B 7.4;< 4.5?7 <.B<6 7:.
Pea, Area )ample Amount 1ppm2
!or Pea, 7
;?<;.@45
;?.<;@4
4B.7@5:
4@.;@;5
45.<<6<
@;B.6?4
7:: !or Pea, 4
[email protected]:7 7::
!or Pea, <
4@;@.;57 7::
!or Pea, 6
45<<.6<: 7::
!or Pea, 5
@;B6?.4: 7::
=espond !actor
Area
(obile Phase =atio
;4.7BB< ;47B.B<66 45.@5:? 45@5.:?4; 46.@75B
[email protected]; 44.:B45 44:B.45@B 65;.5:5: 65;5:.5:: )ample I3* )tandard mixture 17:: ppm2
;: H4 * <: AC>
=espond !actor 1=!2
Pea, Area
)ample Amount 1ppm2
!or Pea, 7
;47B.B<66
;4.7BB<
45.@5:?
46.@75B
7:: !or Pea, 4
45@5.:?4; 7::
!or Pea, <
[email protected];
=etention Time 1minute2 7.:@@ 7.7;? 7.?@? 4.@66 5.?4<
7:: !or Pea, 6
44:B.45@B
44.:B45
65;.5:5:
7:: !or Pea, 5
65;5:.5:: 7::
!= D=A3I9>T 9L0TI>* i2 =espond !actor
!irst injection
Area
(obile Phase =atio
;B.@@;5 ;B@@.;5
;: H4 * <: AC>
=etention Time 1minute2 7.7
Pea, Area )ample Amount 1ppm2
!or Pea, 7
;B@@.;5
;B.@@;5
4@.?B7B
46.7;:5
44.6;7B
44B.:<66
7:: !or Pea, 4
4@?B.7B4@ 7::
!or Pea, <
467;.:544 7::
!or Pea, 6
446;.7B;; 7::
!or Pea, 5
44B:<.66: 7::
ii2
)econd injection
=espond !actor ;6.46<: 45.@?@: 44.7457 47.5B6< 466.<5@6
Area ;646.<:<4 45@?.@:BB 4474.57:? 475B.6<<5 466<5.@6:
(obile Phase =atio
;: H4 * <: AC>
=etention Time 1minute2 7.7:B 7.46< 4.44B 4.;7? 6.:B@
)ample I3* )tandard mixture 17:: ppm2 =espond !actor 1=!2
Pea, Area )ample Amount 1ppm2
!or Pea, 7
;646.<:<4
;6.46<:
45.@?@:
44.7457
47.5B6<
466.<5@6
7:: !or Pea, 4
45@?.@:BB 7::
!or Pea, <
4474.57:? 7::
!or Pea, 6
475B.6<<5 7::
!or Pea, 5
466<5.@6: 7::
)ample I3 17::ppm2 Caffeine Acetone Phenanthrene (ethyl #en/oate Phenatole =espond !actor 1=!2
=espond !actor @:.:@4; 6;.?:@< <7.?@6< 777.;;7 7B;.74;
Area
(obile Phase =atio
@::@.4;:5 6;?:.@<;; <
[email protected]
;: H4 *<: AC>
Pea, Area )ample Amount 1ppm2
!or Caffeine
@::@.4;:5
@:.:@4;
6;.?:@<
7:: !or Acetone
6;?:.@<;; 7::
=etention Time 1minute2 7.:;4 7.7?; 4.;7? 4.:76 @.767
!or Phenanthrene
<
[email protected]
<7.?@6<
777.;;7
7B;.74;
7:: !or (ethyl #en/oate
777;;.7:: 7::
!or Phenatole
7B;74.;:: 7::
DISCUSSION
3uring this experiment a High Performance Li$uid Chromatography 1HPLC2 Agilent D7<76A e$uipped with 0E detector 5 Fm =e"erse Phase C7B column and 4: Fl sample loop was used. At flow rate 7.5 ml 8 min and detector wa"elength at 456 nm the mobile phase ratio 1"8"2 was set at 5: water and 5: acetonitrile at the beginning in order to analy/e and obser"e the effect of mobile phase on LC separation. After all the standard samples which is standard mixture caffeine acetone phenanthrene methyl ben/oate and phenatole were injected the ratio was changed to ;:*<: respecti"ely on the same mobile phase. #ased on the actual procedure from this experiment we need to identify the components contained in the standard mixture by using the optimi/ed LC conditions getting from the abo"e ratio of the mobile phase as well as we should perform a gradient elution separation to impro"e the efficiency of the column. (eaning that isocratic elution is performed with a single sol"ent or constant sol"ent mixture. If one sol"ent does not pro"ide sufficiently rapid elution of all components then gradient elution can be used. In this case increasing amounts of water are added to acetonitrile to create a continuous gradient.
#ut the result shows all the pea,s from the injection process to the sample loop were not separated well. In a re"ersed'phase separation the strength of eluent decreases as the sol"ent becomes more polar. Acetonitrile has high eluent strength and all compounds are eluted rapidly. All the pea,s are obser"ed o"erlapping. !rom the result of chromatogram and area calculation we can see that the =esponse !actor for all the standards injected is almost same. It was so difficult to determine the resolution of the pea,s since the pea,s got o"erlap because the mixture is in high concentration. As we ,now the $uantitati"e analysis in separation method depends upon direct relationship between the area under a pea, or pea, height in the chromatogram and the amount of the compound corresponding to that pea, in the analy/ed sample. Therefore each pea, should be totally resol"ed from any neighboring pea,s. A co'elution or other anomalies such as tailing or fronting will distort or obscure the beginning and ending points of the pea,. Another reason there are some factors that contribute to all the problems stated abo"e. The sample must be degassing properly. )ometime when the pressure was not consistent there must be any air bubble in the mobile phase that fluctuant the instrument. Therefore the instrument should be purge to let the pressure stable. (obile phase that is too cooled also effect the pressure. The 456nm is the most suitable wa"elength because gi"e us "ery nice and sharp pea,. The flow rate or "elocity of the mobile phase is "ery essential in HPLC 1according to the Ean 3eemter 9$uation2. CONCLUSION
The components is identified as well as their pea, retention time.
REFERENCES
7.
),oog Holler and >ierman 5 th 9dition. Principles of Instrumental Analysis. Thomson Learning 7??B
4.
),oog 3.A. -est 3.( Holler !.G. ; th 9dition !undamental of Analytical Chemistry
3.
Saim, N., Tajuddin, R., & Saaid, M. (2014). Analytcal separaton mehods laboraory guide. Selangor: UiTM Pre.
(EXPERIMENT 2) HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC): METHOD DEVELOPMENT
NAME:
NURUL HAZIQAH BINTI HASAN
STUDENT ID:
20166662
PARTNERS! NAMES: 1"
NUR AININA BINTI MOHAMED AINI
2"
NURUL #UNAIDAH BINTI TERMIZI
$"
RAHAYU BINTI ABDUL RAHMAN
DATE OF SUBMISSION:
0%&06&201
