High Performance Liquid Chromatography of Some Analgesic Compounds An Instrumental Analysis Experiment Paul Haddad', Stephen Hutchins, and Michael Tuffy Department of Analytical Chemistry, University of New South Wales, P.O. Box 1, Kensington, New South Wales, Australia 2033
The technique of High Performance Liquid Chromatography (LC) is a useful method for a wide variety of analyses and may be used to complement the older technique of gas chromatography. Much of the work currently puhlished on LC is, however, not directly suitable to illustrate the wide variety of procedures which may he used to enhance the separation characteristics of a mixture. while a t the same time. prove interesting and relevant as an undergraduate experiment. Previously published student experiments on LC include analysis of essential oils ( I ) , analysis of urinary compounds (21, and liquid chromatography with electrochemical detection (3).These experiments provide interesting examples of the auulication of LC but are not desirned to illustrate the effects such fundamental variables as'iolvent composition, pH, etc. In this paper we present an experiment which demonstrates the techniques of solvent selection, gradient elution, pH control, and ion-pairing in the analysis of an analgesic mixture using reversed phase LC on an octadecylsilane (ODs) column. Although this experiment has been developed using fairly sophisticated apparatus, the majority of the procedures described are directly applicable to inexpensive, isocratic instruments. Selection of a suitahle erouu of solutes for the
..
selected compounds are shown in Figure 1. Experimental instrumentation and Reagents T h e liquid chromatograph used consisted of a Waters Assoc. (Milford, MA, USA) Model 6000 solvent pump, Model M45 solvent pump, Model 660 solvent programmer, Model U6K injector, Model 450 variablwvaveleneth detector. and an OmniscriheModel B5217-1
system immediately pl.ior to use. A series of common analgesic compounds was studied using the following mixtures prepared by dissolving the indicated amounts in 50 ml of methanol in a volumetric flask. Mixture A consisted of acetylsalicylic acid, 86.0 mg (May and Baker); paracetamal, 5.1 mg (Fluka, A.G. Switzerland); salicylamide, 87.8 mg (BDH); caffeine, 28.1 mg (BDH);phenacetin, 11.4 mg (ADH). Individual solutions of each component at the same concentration used in the mixture were also prepared using methanol as solvent. Mixture B consisted o l concenlrations or acetylsalicylic acid and salicylamide used above in addition lo salicylic acid (Fisher Chemi-
' Author to whom correspondence should be addressed.
166
Journal of Chemical Education
NHCOCH3
Q
F3txx" $Hz
CHI
C2H5
(dl
le)
CHI
0
If1
Figure 1. Structures of compounds used in this experiment. (a1Salicylic acid. (b) Acetylsalicylic acid. (c) Salicylamide. (dl Paracetamal, (el Phenacetin. If1
Caffeine.
cals) which was included because of the likelihood of its presence as an impurity in aspirin. The ion pairing reagent was "Unichrom" t e t ~ rahutylammonium phosphate (Ajax Chemicals, Sydney); this reagent is equivalent to Waters PIC A (Milford, MA, IJSA). Phosphate buffers (0.025M) were prepared by mixing appropriate amounts of AR NaH2POn and NanHPOa and their p H was adjusted to t h e required values using NaOH or orthophosphoric acid.
Chromatographic Conditions Separation was accomplished using a Chromatographic Systems C18 column (25 cm X 4.6 mm ID). In all cases, 7MIof the test solution was inieeted Rom a 25 ul svrinee. the mobile ~ h a s eflow rate was 2.0 milmin and the detector was operated a t 254 nm with a sensitivity of 0.4 AUFS. T h e recorder char1 speed was 2 cmlmin and all separations were carried out a t 20DC.
Procedures T h e experiment was divided into the sections described below. An indication of the time required to complete each seelion is also given. Soluent Selection. [Requires appmx. 3 hr]. Solvents were prepared with the following compositions: 30,35,40,45,50, and 55% methanol in 0.025M p H 7 phosphate buffer and filtered through a Millipore filter before use. T h e column was allowed to equilibrate when t h e solvent was changed hy flushing with the new solvent for 15 min a t a flow rate of 2.0 mllmin. After this time, 7 ~1 of mixture A was injected and the peaks identified by subsequent injection of the i n d i ~ vidual solutes. T h e technique of gradient elution was demonstrated by injecting mixture A using the following conditions: initial solvent composition 1585 Me0H:pH 7 buffer, final solvent composition 60:40 Me0H:pH 7 buffer, convex gradient (numher 3 on Waters Model 660 Solvent Programmer) using a run time of 12 min. pH Effects [Requires apprux. l1I2hr]. Solvents were prepared with
.
"
.
(TRAP) reagent diluted in accordance with the directions and cam-
hined with methanol to give a solvent composition of 4555 MeOH: TBAP. The pH of the diluted aqueous TBAP reagent was determined to he 7 6 . Fur comparison purposes, a solvent not containing TBAP was prepared using pH 7.6 phosphate buffer mixed with methanol in the same proportions a s above. Mixture B ( I p1) was injected using both of the above solvents, and the peaks were identified by comparison with chromatograms of the sin:,le solutes obtained using the same solvents. Results and Discussion
Solvent Selection
The retention of a solute is expressed as its capacity factor, k', calculated according to the expression
where t , is the retention time of the solute peak, and t o is the retention time of an unretained peak (usuallv the solvent in which the solute is dissolved). Capacity factors for each component of the mixture obtained using the various solvent compositions are listed in Tahle 1,together with the time required to elute the mixture. Capacity factors obtained with other columns may vary slightly from those shown in the table. Table 1.
Capacity Factors for Some Analgesic Compounds Using Different Solvent Compositions
%MeOH Solute Acetylsalicyiicacid Paracetamol Salicylamide Caffeine Phenacetin Analysis time (mln)
30
35
40
45
50
55
0.2 0.6 2.4 4.3 8.3
0.1 0.5 1.8 2.8 5.6
0.1 0.4 1.6 2.3 4.3
0 0.3 1.0 1.4 2.6
0 0.3 0.7 1.1 1.8
0 0.3 0.7 0.9 1.4
8
6
5
4
16
11
Table 1illustrates the effect of variation of solvent polarity on the retention of solutes in reversed phase LC. As the percentage of the organic modifier methanol is increased, the capacity factors decrease, and the analysis time also decreases. The binding of a solute to the covalently bonded hydrocarbon chains of the reversed phase column has been theoretically described by the "solvophohic theory" ( 5 )and is essentially due to solvent effects. The most notable property of the solvent is its surface tension and the retention of an un-ionized solute increases as the surface tension of the solvent increases. The mechanism of the binding process is described in detail elsewhere (6). Since an increase in the ornanic - solvent content of hydro-organic mixtures results in a concomitant decrease in surface tension, then a decrease in retention will also result. This trend is reflected in the results shown in Table 1. A further consideration is the resolution between adjacent peaks. It can be seen from the capacity factors in ~ a b l ethat l resolution between acetylsalicylic acid and paracetamol and also between salicylamide and caffeine is unacceptable for methanol concentrations of 50% and 55%. Conversely, low methanol concentrations give good resolution but unacceptably long analysis times. The optimum solvent is 45:55 Me0H:vH 7 buffer. and the chromatozram of this mixture is " shown as Figure 2. The resolution between ~ e a k shown s in Fieure 1 can be greatly improved without a large corresponding increase in analvsis time through the use of ~ r a d i e n telution. In this technique, the composition of theumobile phase is varied continuouslv during the chromatoeravhic sevaration. Use of a low methanol concentration initizlywith a gradual increase in methanol concentration enables baseline resolution to he achieved, as shown in Figure 3. An interesting feature noticeable in Figure 3 is the appearance of an impurity peak which was identified as salicylic acid. If salicylic acid were deliberately included as a component of the mixture, then separation of the six components could be achieved in an acceptahle analysis time only hy the use of gradient elution. The sequence of elution of the compounds used in Figure 3 can be rationalized in terms of their structures. shown in
Conditions Solvent: MeOHIpH 7 phosphate buffer. Cis column, flow rate: 2.0 rnLirnin, 7 pl iniecfions of mixture A, sensitivity 0.4 AUFS.
INJECT
i
Fisure 2. Separation of analoesic mixture A usina the nntirnm "~ ~7~ . snlvmi miritlrp I-Acetylsalicylic acid: BParacefamol; 3-Saiicyiamide; 4Gaffeine; 6Phenacetin. Conditions: 7 &I injection. mobile phase 4565 Me0H:pH 7 phosphate buffer, tiow rate 2.0 miimin. sensitivity 0.4 AUFS. ~
Figure 3. Separation of analgesic mixture A using gradient elution. 1-Salicylic acid imouritv: . . 2-AceNisalicvlic . . acid: 3-Paracetamol: 4-Salicvlamide: 5Gaffeine: o - P ~ e 1 3 ~ eConzrors ~n 7 " 1 n e c l 3 n mo: c i l , r ~ .1 ~ c : i l . ~l l m i 1: (I. WOt ' : .I+ f namconat ons 6. 4C MeOm'r? 7 3.ller ~ 0 n . gaaen. e ~ r .r.e 3 on .Aa!eri 66( so e n - gwqr?mm?' r-n !.me I > 11 n. 1 :n llle > L miimin. sensitivity 0.4 AUFS
I)'.
Volume 60
Number 2
February 1983
167
1
3
2
n
Table 2.
INJECT
Capacity Factors of Some Analgesic Compounds Obtained at Various Values of pH
1 pH
3
5
7
9
0.8
Solute
Fiaure 4. Seoaration of acidc camoaunds u i n a ion "air chrnmatooranhv 1-
7.6, flow rate 2.0 mllmin. sensitivity 0.4 AUFS
Acetylsallcyi~cacid Paracetamoi Salicylamide Caffeine Phenacetin
0.3 1.0 1.3 2.4
0 0.2 0.9 1.2 2.2
0 0.2 09
0 0.2 0.2
1.2 2.3
2.0
Anaiysis tlme (mini
6.5
6
6
5
Cond~I;ons.Solvent 4555 Me0H:Buffer Clsco1umn. flow rate 2 mlim~n.7 pl injections of mxture A, sensitivity 0.4 AUFS.
Table 3.
either neutral in character or are very weak acids or bases. As such, these compounds would he uncharged a t p H 7 and their retention order results from differences in their hydrophobic character. Effect of pH Capacity factors for the components of mixture A obtained a t several pH values using 4555 MeOKbuffer as solvent are listed in Table 2. As the p H is decreased, retention increases for the acidic compound acetylsalicylic acid due to partial suppression of its ionization a t pH 3. The technique of using pH adjustment to manipulate the retention of acidic or basic solutes is known as "ion-suppression." The effects of pH are further illustrated by reference to the retention of salicylamide. Salicylamide, being a very weak acid (pK, = 8.371, shows constant retention from pH 3-7 indicating that in this pH range, it is un-ionized. However.. a t . nH 9. nartial ionization occurs resulting in a decrease in retention. The other species in the mixture are too weak in their acid-base properties to show appreciable p H effects. The impurity, salicylic acid (not listed in Table 21, co-elutes with acetylsalicylic acid near the void volume except a t pH 3, where it elutes a t a capacity factor of 0.3. However, this retention correswonds to that of waracetamol, so salicvlic acid and paracetarnil co-elute a t p ~ 3 . I t can be concluded from these results that the optimum separation of the major components is achieved a t p H 7, using the solvent 45:55 Me0H:pH 7 buffer (Fig. 1).
-
lon-pair Chromatography Ion suppression methods are not applicable to the LC of moderately strong acids or bases since the pH values required to prevent ionization of these species are too extreme for reversed phase columns. The pH of the mobile phase should be limited to the ranee 2-9 due to dissolution of the silica suvvort a t high pH and cleavage of the bound hydrocarbona&us chain a t low DH.It is uossihle however to sewarate ionic s~ecies by reversedbhase LC using the technique of ion-pairchromatography. Here the pH is adjusted so that the solute is fully ionized and an ionic alkyl compound is introduced into the mobile phase. This effectively neutralizes the charge on the ionized solute, and retention occurs. The mechanism operating in ion-pair chromatography is under intensive debate and the theories nrooosed have been recentlv reviewed (71. For LC of acidic species, a cationic alkyl cokpound is iskd as the ion pairing reagent and tetraalkylammonium salts are frequently employed. Such a compound is tetrabutylammonium phosphate (TBAP), which carries an appropriate charge and has sufficient lipophilic character to assist in the retention of solutes by reversed phase LC.
168
Journal of Chemical Education
10
Capacity Factors of Some Acidic Compounds in the Presence and Absence of Ion Pairina Reaaent
Solvent Solute
1
2
Acetylsalicylic acld Salicylic acid Salicylamide
0 0.7
0.3 1.1 1.4
Analysis time (min)
2.3
3.4
0
Conditions. Solvent 1 , 45:55 Me0H:pH 7.6 phosphate buffer: solvent 2. 4555 MeOH: TSAP, flow rate 2.0 miimin, 6~~column. 7 PI njectans of mixture B, sensit~vity0.4 AUFS.
To examine the technique of ion-pair chromatography, mixture B (consisting of the acidic species salicylic acid, acetylsalicylic acid, and salicylamide) was injected using 45% MeOH solvent containing either tetrabutylammunium phosphate (TBAP) reagent (at pH 7.6) or pH 7.6 phosphate buffer. The results are shown in Table 3. At pH 7.6 and in the absence of the ion pairing reagent, both acetylsalicylic acid and salicylic acid are ionized and elute together a t the void volume. Salicylamide is only very slightly ionized and shows some retention. When TBAP reagent is added with the vH unchaneed. all swecies show increased retention in accordance with-predicted behavior and i t is now wossible to resolve the mixture without further alteration of solvent composition. The chromatogram of the mixture obtained with the use of ion pairing reagent is shown in Figure 4. Conclusions
The experiment described provides an example of the effects of variation of solvent parameters commonly employed in LC, together with an interpretation of the results. The equipment used was fairly sophisticated; however, much of the experiment could be easily adapted to more simple instrumentation. Possible extensions to the experiment include quantitative studies on analgesic mixtures and the effect of adding a salt (e.g., NazSOJ to the mobile phase. Provided the solvents to he used are prepared and filtered in advance, the experiment can be completed in approximately 5% hr. Literature Cited !I1 McKcme, H. T., J . C H E M E D O C56. 698 11979). (21 B ~ S ~D.I . MIII~~. H. L.. wulline.A. G..senrtlrber, F. c., veening,H. z.,.I. (:HEM R I ~ U C . . ~768 ~ , (1977). (31 Kssingei. P.T.. Fehco. L. I. Kmg, . W.1'. Prrhlr. I.. A , Riggln. R. M..and Sliuup, H.
w.,
