EXPERIMENT 2: HIGH PERFORMANCE LIQUID CHROMATOGRAPHY CHROMATOGRAPHY (HPLC) : METHOD DEVELOPMENT
NAME
:
MUHAMMAD RAHIMI BIN ZAHURI
( 2 01 58 2 8! ! )
PART PARTNER NER"# "# NAME NAME
:
A$MAL A$MAL AR#Y AR#YA AD BIN BIN MOHD MOHD RAFFI RAFFI
(20158% (20158%5&0 5&0&) &)
MOHD IQBAL BIN NORAZMAN
(20158&012)
MUHAMMAD RAHIMI BIN ZAHURI
(2015828!!)
#YARAFUDDIN #YARAFUDDIN BIN MOHD #HAM#IBY
(20158%288)
DATE OF EXPERIMENT: DATE OF #UBMI##ION OF REPORT:
Introduction: There are three objectives of this experiment. Firstly, the objective is to study the development for optimizing the separation of mixture of five compounds which are standard mixtures of caffeine, acetone, methyl benzoate, phenatole, and phenanthrene using !"# by varying the mobile phase composition. $econd, to identify the individual components exist in the mixture and third is to optimize the separation between each pea%s using gradient elution method. !"# is the most widely used of all of the analytical separation techni&ue. This type of instrument is suitable for the separation of nonvolatile compounds. For this experiment, reverse'phase is used at which stationary phase is non polar while the mobile phase is polar. This will ma%e the most polar component to be eluted first. In the li&uid chromatography, it is more complicated due to the interaction of the components of analyte with both the stationary phase and the mobile phase. (y that, increasing the mobile phase polarity will increases the retention time of the components. The polarities of various analytes functional groups in increasing order are as shown below: ydrocarbon ) *ther ) *ster ) +etones ) ldehydes ) mides ) mines ) lcohols -ater is the most polar compounds among the compounds containing any of the preceding functional groups. In this experiment, compounds which are caffeine, acetone, methyl benzoate, phenatole, and phenanthrene are determined by varying the mobile phase composition throughout the analysis. This is %nown as gradient elution techni&ue. /radient elution is a condition at which the composition of the mobile phase is changed during the separation. Two or more solvents that differ in polarity are employed. The method was intended to decrease the retention time for the later eluting components so that they can elute faster which then produces narrower pea% thus improves the pea% shape. This experiment uses water0acetonitrile gradient at which more hydrophobic components will elute when the mobile phase consists of mostly acetonitrile which gives a relatively hydrophobic mobile phase. The more hydrophilic compounds will elute under conditions of relatively low acetonitrile and high water.
*xperimental: Instrument used: "i&uid chromatograph 1gilent /2324 !"#5 e&uipped diode array detector 1665 and 7m 8! #29 column and ;7" sample loop. a. Instrument set up 6etector wavelength : 4nm Flow rate : 2.m" min'2
8esults and 6iscussion:
8esolution 1Isocratic *lution5 :? 1;:; v:v5 !ea%
#alculation
−0.978 ) =¿ 0.0551 + 0.0681
8esolution, 8s
(
2 1.126
2'
−1.126 ) =¿ 0.0551 + 0.1068
.4;
(
2 3.251
'3
−3.251) =¿ 0.1548 + 0.1068
@.3;
(
2 5.552
3'4
−5.552 ) =¿ 0.5191 + 0.1548
2=.A
(
2 20.585
4'
44.@2
Table 2.2 8esolution 8un 2
:? 1=;:3; v:v5 !ea%
#alculation
−0.957 ) =¿ 0.0591 + 0.0520
8esolution, 8s
(
2 1.057
2'
−1.057 ) =¿ 0.0650 + 0.0520
2.9;
(
2 1.736
'3
−1.736 ) =¿ 0.0650+ 0.0837
22.@2
(
2 2.311
3'4
−2.311) =¿ 0.2342+ 0.0837
=.=3
(
2 5.173
4'
29.;;
Table .2 8esolution 8un 2 !ea%
#alculation
8esolution, 8s
(
−0.959 ) =¿ 0.0496 + 0.0558
2 1.059
2'
−1.059 ) =¿ 0.0600 + 0.0496
2.A;
(
2 1.740
'3
−1.740 ) =¿ 0.0753 + 0.0600
2.43
(
2 2.315
3'4
−2.315 ) =¿ 0.2192 + 0.0600
9.;
(
2 5.166
4'
;.4
Table . 8esolution 8un
#omponent
8etention Time
-idth
#affeine
;.A@;
;.;@24
cetone
2.;A
;.;23
2.=3A
;.;@
!henatole
.32
;.;==A
!henanthrene
.2=
;.23;=
Table 3.; 8etention time for individual component
8esolution 1/radient *lution5
!ea%
#alculation
−0.981 ) =¿ 0.0714 + 0.0540
8esolution, 8s
(
2 1.128
2'
−1.128 ) =¿ 0.0531 + 0.0540
.34
(
2 2.417
'3
−2.417 ) =¿ 0.0550 + 0.0531
4.;=
(
2 2.799
3'4
−2.799) =¿ 0.1448 + 0.0550
=.;=
(
2 4.016
4'
Table .; 8esolution for /radient *lution
6iscussion:
2.29
In the experiment, a igh "i&uid !erformance #hromatography 1!"#5 gilent /2324 was used. The detector used was diode array detector 1665 which included 7m 8! #29 column and ;7" sample loop. !"# is a method or techni&ue to separate, identify, and &uantify the components in a mixture. It is suitable for nonvolatile compounds, thermally unstable, and has high molecular weights. t the flow rate of 2.m" min '2 and the detector of 4nm, the mobile ratio was set up at ;B acetonitrile and ;B water at the beginning of the experiment in order to analyze the effect of mobile phase composition on the chromatography separation. fter the standard mixture is injected, the process is run and the pea% obtained is analyzed. Then the ratio was changed to =;:3; respectively on the same mobile phase. The resolution of the chromatogram is calculated by using the formula stated below:
:
R
=
( −t 1 ) W 1 + W 2
2 t 2
-here t is the retention time and - is the pea% width. From the experiment, we need to identify the components contained in the standard mixture by using the optimized li&uid chromatography conditions getting from the ratio of the mobile phase. ?ther than that, gradient elution separation must also be conducted to improve the efficiency of the column. This means that if the isocratic elution does not provide sufficiently rapid elution of all the components, then gradient elution can be used in order to replace the method. From the chromatogram obtained with the resolution calculated, it can be said that by comparing the average resolution for both mobile phase compositions, we can see that the mobile phase ratio of =;B acetonitrile and 3;B water gives the best resolution which is 2.9;. This indicates that the resolution is good and the efficiency of the separation increases. :water ratio. This experiment also identified the components in the standard mixture when it is injected individually by using the optimum mobile phase composition with the rati of =;:3; #>:water. This ratio is used due to the best composition baseline separation compared to the ;C; ratio. The compound in the standard mixture is identified by comparing the retention time of the standard mixture with the retention time of the individual compound. From the chromatogram
obtained, it indicates that pea% 2 is caffeine, followed by pea% which is acetone, pea% 3 is methyl benzoate, pea% 4 is phenatole, and pea% is phenanthrene. From the chromatogram obtained from both of the methods which are isocratic elution method and gradient elution method, it can be compared that there are some differences from the condition of separation. In isocratic elution, the pea% width increases as the retention time increases. This leads to late eluting pea%s which is broad.
#onclusion: From the experiment, it can be concluded that by varying the mobile phase composition in !"#, the method development of optimizing a separation of a standard compounds can be determined.
8eference:
(n.d.). Retrieved from HIGH PERFORMANCE LIQUID CHROMATOGRAPH !
HPLC" #tt$"%%&&&.'#emide.'o.*%+n+,-i%'#rom+tor+$#-%#$,'.#tm, (n.d.). Retrieved from Hi# $erform+n'e ,i/id '#rom+tor+$#-" #tt$"%%'#em&i*i.'d+vi.ed%An+,-ti'+,0C#emitr-%Intrment+,0An+,-i %C#rom+tor+$#-%Hi#0$erform+n'e0,i/id0'#rom+tor+$#1+im2 N.2 T+3ddin2 R.2 4 1++id2 M. (5678). E9$eriment 7 G+ C#rom+tor+$#-(GC)" O$timi:+tion of F,o& R+te +nd Co,mn Tem$er+tre. In ANALYTICAL SEPERATION METHODS LABORATORY GUIDE 2nd edition ($$. 7!5). 1e,+nor" UiTM Pre. 1*oo2 ;. H. (5666). An+,-ti'+, C#emitr-" An Introd'tion2
