5- NORMAL PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC DETERMINATION OF CHLORPHENOXAMINE HYDROCHLORIDE, CAFFEINE AND 8-CHLOROTHEOPHYLLINE
Y. M. Dessouky, H. H. Hassanein, M. Abdul-Azim Mohammad* and R. S. Hanafy** Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasrel-Aiani, 11562, Cairo, Egypt. Received:14-3-2004 Accepted:16-5-2004
Abstract: INTRODUCTION An isocratic, rapid and reproducible high performance liquid chromatographic (HPLC) Chlorphenoxamine hydrochloride (CPHX), [I] method for quantitative determination of chlor- is a histamine H1- receptor antagonist with anti- phenoxamine hydrochloride (CPHX), caffeine muscarinic properties 1. Caffeine (CAFF), [2] is a (CAFF) and 8-chlorotheophylline (CTHE) has been xanthine derivative with CNS stimulant activity 1-3. adopted. The optimal conditions for separation and 8-Chlorotheophylline (CTHE) [3] is also a xanthine quantitative determination of these compounds derivative that forms with dipenhydramine a were a normal phase mode chromatography and compound named dimenhydrinate that is widely isocratic elution at ambient temperature. A silica used for alleviation of motion sickness 1-3. column and a mobile phase consisted of ethyl Combination of the three drugs is known 1,4. acetate - methanol (50:50 v/v) / triethylamine pH 9, CPHX was determined by titrimetric 2, have been used at a flow rate of 1 ml min-1. The electrochemical 5, spectrophotometric 6-8, derivative components of column effluent were monitored by spectrophotometric 9,10, derivative ratio spectro- 11 11 UV detector at 254 nm. The retention times (tR) for photometric , chemometric and spectro- CPHX, CAFF and CTHE were 6.15, 4.23 and 2.97 fluorimetric 12 methods. Chromatographic methods min, respectively. The linearity range for CPHX, including HPLC 13, TLC 14 and GC15,16 were also CAFF and CTHE were 37.5 - 725 µg ml-1, 0.75 - 48 reported. CAFF was determined in µg ml-1 and 0.1 - 40 µg ml-1, respectively. The mean pharmaceutically dosage forms and also in accuracy ± % RSD for CPHX, CAFF and CTHE biological fluids by titrimetry 3,17,18, 19-21 were 100.06 ± 0.47, 99.43 ± 0.60 and 100.65 ± spectrophotometry , derivative spectrophoto- 10,22 1.25, respectively. The results obtained by the metry , derivative ratio spectrophotometry 11,23,24 11,25,26 27 recovery studies indicated suitable validation , chemometry , near IR spectrometry 28 parameters. The proposed method showed good and NMR spectrometry . Chromatographic efficiency for simultaneous quantitative methods were also reported, such as HPLC determination of CPHX, CAFF and CTHE in bulk 2.13,23,24,29-35, TLC 14,36,37, GLC 38,39and GC 40,41. and tablet dosage from. Statistical comparison CTHE was determined by titrimetry 3,4,42, between the results obtained by the proposed conductometry 4,43, spectrophotometry 44, HPLC method and those obtained by the selected official 13,35, and TLC 14. methods for these drugs was carried out and no significant differences were found. Cl Keywords :chlorphenoxamine, chlorotheophylline, caffeine,pharmaceutical analysis, HPLC. O CH N 3 * Postal address: Faculty of Pharmacy, Cairo CH CH university- Kasrel-Aini- 11562-Cairo-Egypt. 3 3 ** Faculty of Pharmacy, Cairo university, Beni- Suef Branch.
[1] Chlophenoxamine
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O CH • Ethyl acetate for HPLC (Panreac, 3 Barcelona, Spain). H3C N • Triethylamine (Fluka-Sigma-Aldrich, N Germany). • Reference standards: CPHX, CAFF and O N N CTHE were obtained from the Egyptian International Pharmaceutical Industries CH Co.(EIPICO), 10th of Ramadan City, 3 Egypt. Purity of these standards was 99.82, 99.65 and 99.76% for CPHX, CAFF and [2] Caffeine CTHE, respectively as certified by the dealer. The purity was also checked by the pharmacopoeial methods. R O • Emeral tablets , batch number 020697 H from the Egyptian International Pharmaceutical H3C N N Industries Co. (EIPICO), 10th of Ramadan Cl City, Egypt. It is labeled to contain 30 mg, N 50mg and 20 mg per coated tablet for O N CPHX, CAFF and CTHE, respectively. CH 3 Chromatographic condition The mobile phase consisted of ethyl acetate- [3] 8-Chlorotheophylline methanol (50:50 v/v), adjusted to pH 9 using triethylamine was flowing at a constant flow rate of A pharmaceutically combination dosage forms 1.0 ml min-1. A Shimpack NP- Sil. column (150 of CPHX, CAFF and CTHE used to treat nausea, mm x 6.0 mm i.d., 5 µm) was used as stationary vertigo and vomiting are available on the market. phase. Detection was carried out using an UV The only two stated methods for the determination detector set at 254 nm. of these drugs in mixture are a reported TLC- 14 densitometry and an HPLC manufacturer's Standard preparations 13 procedure . Thus, the purpose of this study was to 1) Stock standard solutions: develop rapid, easy and precise liquid Standard stock solutions of 5000 µg ml-1 CPHX, chromatographic (HPLC) method for the 500 µg ml-1 CAFF and 200 µg ml-1 CTHE were simultaneous determination of the three drugs. prepared by dissolving 250 mg standard CPHX, 25 mg standard CAFF and 10 mg standard CTHE each EXPERIMENTAL in 50 ml ethyl acetate- methanol (50:50 v/v).
Instrumentation and Parameters 2) Working standard solutions: A liquid chromatographic system: Shimadzu Each standard stock solution (20 ml) was class Avp Interface Module (Shimadzu-Japan) diluted to 100 ml with the same solvent system. equipped with: This give a concentration of 1000 µg ml-1 CPHX, • An isocratic HPLC pump (Model LC-10 ADVP, 100 µg ml-1 CAFF and 40 µg ml-1 CTHE. These Shimadzu, Japan). solutions were used as the working standards for • UV-visible detector (Model SPD-10A- VP, the analysis. Shimadzu, Japan). • A Rheodyne syringe loading sample injector 20 Laboratory prepared mixtures: µL (Mode l7725i, Catati, California,USA). Accurately weighed 15, 150 mg standard • Column: Shimpack NP-Sil. (150mm x 6mm i.d., CPHX, 25, 250 mg standard CAFF and 10, 100 mg 5µm) , (Shimadzu, Japan). standard CTHE were separately dissolved each in • Ultrasonic Processor Crest, 20 KHz frequency. 100 ml ethyl acetate- methanol (50:50 v/v). This give concentrations of 150, 1500 µg ml-1 CPHX, 250, 2500 µg ml-1 CAFF and 100, 1000 µg ml- 1 Reagents and Chemicals CTHE, respectively. Different aliquots of these • Methanol for HPLC (Riedel de Häen, Sigma- solutions within the linearity ranges of the studied Aldrich, Germany). drugs were mixed together into a series of 1-ml
54 Bull. Fac. Pharm. Cairo Univ., Vol. 42, No. 1 (2004)
Eppendorf centrifuge tubes and used as the determination of the studied drugs might be a great laboratory prepared mixtures. need.
Sample of pharmaceutical preparation (tablets): Method Development Twenty tablets were weighed, ground to fine The challenge encountered in designing the powder and thoroughly mixed. A quantity of the chromatographic conditions during this study was powder equivalent to one tablet was accurately the separation of CAFF and CTHE, because they weighed in a 50-ml volumetric flask, then 35 ml of have more or less similar structure and polarity. A the solvent system, ethyl acetate- methanol (50:50 previously reported HPLC method for estimation of v/v) were added. The mixture was sonicated for 20 CTHE in the double salt (dimenhydrinate) with min, then cooled, completed to the mark with the diphenhydramine, a congener of chlorphenoxamine same solvent and filtered using 0.45 µm Millipore employed a gradient elution mode 2. Another filter. The first 10 ml of the filtrate was rejected and reported HPLC method for the analysis of CAFF the rest was used for the analysis. and CTHE in mixture with diphenhydramine applied an acidic mobile phase and RP-8 column Procedure and linearity because it has one of the lowest hydrophobic and Accurately measured aliquots of working silanol activity factors35. The presence of silanol standard solutions equivalent to 37.5 - 750 µg, 0.75 blocker such as triethylamine facilitates elution of - 48 µg and 0.1 - 40 µg of CPHX, CAFF and basic drugs without tailing 45. In addition, adjusting CTHE, respectively were transferred into three pH of the eluent was needed to allow differences in series of 1-ml Eppendorf centrifuge tubes and the the degree of ionization, change elution order and volume was brought to 1 ml with the same solvent. modify resolution 35. A volume of 20 µl of each solution was separately On the other hand, silica columns used with non injected into the chromatograph. The peak area of aqueous eluents such as methanol containing ionic each drug concentration was recorded. A calibration modifier provide stable and flexible systems for the graph for each drug was obtained by plotting peak analysis of drugs by HPLC 46. Retention on silica areas against its corresponding drug concentrations. column / non aqueous ionic eluent systems is The regression equations were computed. mediated primarily via cation exchange with surface silanol 47. Assay of laboratory prepared mixtures Different parameters affecting the The laboratory prepared mixtures were chromatographic separation by the proposed analyzed applying the same procedure as under method were studied. Several columns were tested linearity starting from `` the volume was brought and several pH modifiers were tried to cause ……``. The peak areas were recorded and the differences in the degree of ionization of the studied concentration was calculated from the respective drugs and hence, modify their elution order. The regression equation of each drug. composition of the mobile phase was adjusted after varying of the less polar (ethyl acetate) to the polar Assay of pharmaceutical preparation (methanol) ratios and also the nature of the pH Different aliquots of sample preparation within modifier. Meanwhile, increasing polarity and the linearity ranges of the studied drugs were acidity of the mobile phase resulted in bad transferred into a series of 1-ml Eppendorf separation between CAFF, CTHE and tailing of centrifuge tubes. The procedure as mentioned under CPHX peak. Also, at neutral and acidic pH, both procedure and linearity was followed starting from CAFF and CTHE were coincident. On the other `` the volume was brought ……``. Concentration of hand, the increase of pH of the mobile phase using such drug was calculated from each regression drops of the silanol blocker, triethylamine up to pH equation. 9, afforded a good separation and better peak shapes with no tailing. In addition, detection was RESULTS AND DISCUSSION carried out at 254 nm, since at longer wavelengths, weak or no response with CPHX is expected (λmax = The only HPLC method available for analysis of 254 nm). such mixture is a manufacturer's procedure. TLC Finally, equal proportions of polar and less technique may be tedious and time consuming. polar solvents, suitable amount of silanol blocker Because HPLC is probably the most powerful, and basic pH at ambient temperature solely resulted versatile, rapid and selective tool for quantitative in satisfactory results. determination of many individual components in a The retention times are 6.15, 4.23 and 2.97 min mixture in one single procedure, a thought to adopt for CPHX, CAFF and CTHE, respectively as a new HPLC method for the simultaneous presented in figure (1). The system suitability tests
Bull. Fac. Pharm. Cairo Univ., Vol. 42, No. 1 (2004) 55 are used to verify that the resolution and method was done and the results are summarized in reproducibility of the chromatographic system are table VI. adequate for the analysis 2. The results obtained are shown in table I. Specificity and Accuracy: Specificity is the ability of the analytical method Method validation to measure the analyte response in the presence of Linearity: interferences. This was checked by analyzing the A linear relationship between peak areas and content of CPHX, CAFF and CTHE in coated drug concentration was obtained in ranges of 37.5 – tablets preparation containing them by the proposed 750 µg ml-1, 0.75 - 48 and 0.1 - 40 µg ml-1 for method. The obtained results are shown in table CPHX, CAFF and CTHE, respectively. The VII, where the low RSD% values indicate that the following regression equations were computed: method is applicable for the determination of these drugs in the pharmaceutical dosage form with no YCPHX = 1392.56 x – 10608.500 (r = 0.9997) interferences. To ensure the accuracy of the YCAFF = 25363.51 x – 9628.260 (r = 0.9999) proposed method, standard addition technique was YCTHE = 28167.14 x – 2092.464 (r = 0.9999) applied and the results are summarized in table VII. The percent recoveries for CPHX were from 99.49 Linear regression analysis for the obtained to 101.00, for CAFF were 98.90 to 100.20 and for calibration curves of the study drugs was done. CTHE from 99.70 to 101.00. Because the cited Table II shows linear regression data obtained and drugs ratio in the tablets preparation were not in the main validation parameters. Relative standard agree with the obtained linearity ranges of these deviation (RSD %) of slopes ranged between 0.13 - drugs, repeated injections would be essential. Both 0.43 for the three compounds in standards showing CAFF and CTHE could be determined good fit of individual points to the regression line. simultaneously in low concentrations at which the In all cases linearity plots had correlation response of CPHX was weak. Thus, repeated coefficients ≥ 0.9997. injections containing high concentrations of the cited drugs to estimate CPHX were needed. Precision and Selectivity: Different concentrations within the linearity Sensitivity: range of pure CPHX, CAFF and CTHE were The proposed method was capable to determine analyzed by the proposed HPLC method to check CPXH, CAFF and CTHE at low concentration the reproducibility of the results. The mean levels down to 37.5 µg ml-1, 0.75 µg ml-1 and 0.1 µg percentage recoveries ± RSD% were 100.06 ± 0.47, ml-1, respectively. 99.43 ± 0.60 and 100.65 ± 1.25 for CPHX, CAFF and CTHE, respectively as presented in table III. In Conclusion addition, the proposed HPLC method was valid for In comparison with other analytical techniques, the determination of different concentrations within HPLC greatly reduces the analysis time. Besides, the linearity range of pure CPHX, CAFF and CTHE determination of several individual components in laboratory prepared mixtures. The mean present in a mixture in one single procedure can be percentage recoveries ± RSD% were 100.02 ± 0.45, selectively done using HPLC. Owing to these 99.69 ± 0.89 and 99.91 ± 0.88 for CPHX, CAFF merits a rapid, accurate, precise and reproducible and CTHE, respectively. The results are given in HPLC method for the determination of CPHX, table IV. CAFF and CTHE in both pure form and tablet Statistical comparison between the results dosage form was presented. The method is capable obtained by the proposed method and those for simultaneous determination of the three drugs in obtained by the selected official methods showed bulk powder and also in tablet dosage form by no significant differences as given in table V. Intra- repeated injections. Hence, it can be employed for and inter-day precision of the proposed HPLC routine quality control procedures of these drugs.
56 Bull. Fac. Pharm. Cairo Univ., Vol. 42, No. 1 (2004)
Table I: System suitability tests
No. Parameters CPHX CAFF CTHE
1 Theoretical plates 2988 2337 2258 2 Resolution factor (R) 4.80 4.20 3 Selectivity factor (α ) 3.84 2.52 4 Repeatability 0.47 0. 60 1.25 (RSD% of eight injections)
Table II: Linear regression data for calibration curves (peak area vs. concentration) and the main validation parameters
Data CPHX CAFF CTHE
Intercept 10608.50 9628.26 2092.46 Sa* 67.97 26.13 11.27 RSD% of intercept 0.64 0.29 0.19 Slope 1392.56 25363.51 28167.14 Sb** 16.48 104.65 57.96 RSD% of slope 0.43 0.20 0.13 r 0.9997 0.9999 0.9999
* Standard error of intercept. ** Standard error of slope.
Table III: The results of the proposed method and the official methods in the determination of studied drugs in pure form.
Ex CPHX CAFF CTHE No. HPLC method Official method* HPLC method Official method** HPLC method Official method*** (Recovery %) (Recovery %) (Recovery %)
1 99.94 100.73 99.94 99.92 99.95 99.02 2 99.92 100.38 100.03 100.06 99.97 99.26 3 100.08 99.57 98.66 100.24 101.91 100.98 4 99.94 101.10 99.96 99.16 99.89 101.22 5 101.18 100.87 99.00 99.70 100.64 99.59 6 99.65 99.41 99.48 101.25 101.31 100.49 7 99.87 99.65 98.84 8 99.87 98.75 102.70 Mean 100.06 100.34 99.43 100.06 100.65 100.09 %RSD 0.47 0.70 0.60 0.69 1.25 0.93
Each result is the average of three experiments. * U.S.P. (1985): a non aqueous titrimetric method 2. ** B.P. (2001): a non aqueous titrimetric method 3. *** B.P. (2001): a precipitimetric titration method 3, (as a part of dimenhydrinate).
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Table IV: Determination of CPHX, CAFF and CTHE in laboratory prepared mixtures by the proposed HPLC method
Ex CPHX CAFF CTHE % % % No. Taken Found Recovery Taken Found Recovery Taken Found Recovery (µg ml -1) (µg ml -1) (µg ml -1)
1 3.0* 5.0 5.00 100.00 2.0 2.00 100 .00 2 7.5* 12.5 12.28 98.24 5.0 4.99 99.80 3 12.6* 21.0 21.13 100.62 8.4 8.39 99.88 4 15.0* 25.0 24.80 99.20 10.0 10.06 100.60 5 18.0* 30.0 30.12 100.40 12.0 11.80 98.33 6 60.0 60.11 100.18 100.0* 40.0 40.34 100.85 7 150.0 149.93 99.95 15.0 14.85 99.00 20.0 19.81 99.05 8 240.0 241.44 100.60 40.0 40.15 100.38 16.0 16.13 100.81 9 360.0 357.68 99.36 600.0* 240.0* 10 450.0 449.99 100.00 750.0* 300.0* Mean 100.02 99.69 99.91 %RSD 0.45 0.89 0.88 Each result is the average of three experiments. * The value is out of linearity range
Table V: Statistical comparison between the results of the proposed method and Official methods in determination of pure drugs
Value CPHX CAFF CTHE HPLC Official HPLC Official HPLC Official method method* method method** method method***
Mean 100.06 100.34 99.43 100.06 100.65 100.09 S.D. 0.47 0.70 0.60 0.69 1.26 0.93 n 8 6 8 6 8 6 Variance 0.22 0.49 0.36 0.48 1.59 0.86 t (2.179) 0.847 1.787 0.957 F (4.880) 2.227 1.333 1.849
Figures between parentheses represent corresponding tabulated values of t & F at P =0.05 * U.S.P. (1985): a non aqueous titrimetric method 2. ** B.P. (2001): a non aqueous titrimetric method 3. *** B.P. (2001): a precipitimetric titration method 3, (as a part of dimenhydrinate)
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Table VI: Intra- and inter-day variations of CPHX, CAFF and CTHE by the proposed method
CPHX CAFF CTHE
Intra-day
-1 49.98 350.96 650.06 10.01 24.97 44.84 8.04 21.87 35.19 Mean of concentration (µg ml ) n = 3 0.71 0.51 0.45 0.17 0.40 0.62 0.11 0.35 0.35 SD 1.42 0.15 0.07 1.70 1.60 1.38 1.37 1.60 0.99 %RSD
Inter-day
0 Day 50.12 349.97 652.43 10.01 24.84 44.69 8.00 21.90 34.95 -1 Mean of concentration (µg ml ) n = 3 0.74 0.82 0.87 0.19 0.45 0.46 0.17 0.30 0.27 SD 1.48 0.23 0.13 1.90 1.81 1.03 2.13 1.37 0.77 %RSD
1 Day 50.00 350.82 650.31 9.96 25.02 45.27 7.97 22.08 35.07 -1 Mean of concentration (µg ml ) n = 3 0.95 1.06 1.00 0.12 0.43 0.46 0.09 0.35 0.40 SD 1.90 0.30 0.15 1.20 1.72 1.02 1.13 1.59 1.14 %RSD
2 Day 49.83 349.46 649.51 9.92 25.16 44.95 8.01 22.13 34.61 -1 Mean of concentration (µg ml ) n = 3 0.79 1.19 1.11 0.09 0.27 0.41 0.17 0.41 0.34 SD 1.59 0.34 0.17 0.91 1.07 0.91 2.12 1.85 0.98 %RSD
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Table VII: Application of standard addition technique to the analysis of CPHX, CAFF and CTHE in pharmaceutical preparation by the proposed HPLC method.
Ex. CPHX CAFF CTHE No. Taken Found % Added Found % Taken Found % Added Found % Taken Found % Added Found % (µg ml-1) Found (µg ml-1) Recovery (µg ml-1) Found (µg ml-1) Recovery (µg ml-1) Found (µg ml-1) Recovery
1 7.5* 12.5 11.99 95.92 30 29.70 99.00 5 4.76 95.20 6 6.04 100.67
2 9.0* 15.0 14.42 96.13 10 9.89 98.90 6 5.87 97.83 30 29.91 99.70
3 12.0* 20.0 19.43 97.15 20 19.90 99.50 8 7.75 96.88 4 4.03 100.75
4 22.5* 37.5 36.35 96.93 2 2.00 100.00 15 14.63 97.53 16 15.96 99.75
5 24.0* 40.0 38.32 95.80 5 5.01 100.20 16 15.48 96.75 10 10.10 101.00
6 60.0 57.31 95.52 60 60.60 101.00 100.0* 40 38.37 95.93 ------
7 150.0 144.66 96.44 150 150.80 100.53 250.0* 100*
8 240.0 230.65 96.10 400 398.21 99.55 400.0* 160*
9 360.0 343.74 95.48 300 302.49 100.83 600.0* 240*
10 480.0 469.99 97.91 200 198.98 99.49 800.0* 320*
11 600.0 579.00 96.50 ------1000.0* 400*
Mean 96.33 100.28 96.39 99.52 96.69 100.37
%RSD 0.92 0.71 0.63 0.58 1.01 0.61
Each result is the average of three experiments. *The value is out of linearity range.
60 Bull. Fac. Pharm. Cairo Univ., Vol. 42, No. 1 (2004)
Fig 1: HPLC chromatogram of 20 µl injection of synthetic mixture of : : a) 15 µg ml-1 CTHE b) 12.5 µg ml-1 CAFF c) 215 µg ml-1 CPHX
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ﺘﻘﺩﻴﺭﺃﻴﺩﺭﻭﻜﻠﻭﺭﻴﺩﺍﻟﻜﻠﻭﺭﻓﻴﻨﻭﻜﺴﻤﻴﻥ ﻭﺍﻟﻜﺎﻓﻴﻴﻥ ﻭ٨-ﻜﻠﻭﺭﻭﺜﻴﻭﻓﻴﻠﻠﻴﻥ ﺒﺎﺴﺘﺨﺩﺍﻡ ﺍﻟﻁﻭﺭﺍﻟﻌﺎﺩﻯ ﻟﻁﺭﻴﻘﺔ ﻜﺭﻭﻤﺎﺘﺠﺭﺍ ﻓﻴﺎ ﺍﻟﺴﺎﺌل ﺫﺍﺕ ﺍﻟﻜﻔﺎﺀﺓ ﺍﻟﻌﺎﻟﻴﺔ. ﻴﺤﻴﻰ ﻤﺼﻁﻔﻰ ﺩﺴﻭﻗﻰ، ﺤﺴﻨﻴﻥ ﺤﺴﻥ ﺤﺴﻨﻴﻥ، ﻤﺤﻤﺩ ﻋﺒﺩﺍﻟﻌﻅﻴﻡ ﻤﺤﻤﺩ، ﺭﺸﺎ ﺴﻴﺩ ﺤﻨﻔﻰ ﻗﺴﻡ ﺍﻟﻜﻴﻤﻴﺎﺀ ﺍﻟﺼﻴﺩ ﻟﻴﺔ- ﻜﻠﻴﺔ ﺍﻟﺼﻴﺩ ﻟﺔ- ﺠﺎﻤﻌﺔ ﺍﻟﻘﺎﻫﺭﺓ- ﻗﺼﺭ ﺍﻟﻌﻴﻨﻰ١١٥٦٢- ﻤﺼﺭ
ﺘﻡ ﻓﻰ ﻫﺫﺍ ﺍﻟﺒﺤﺙ ﺇﺴﺘﻨﺒﺎﻁ ﻁﺭﻴﻘﺔ ﻟﻠﺘﻘﺩﻴﺭﺍﻟﻤﺘﺯﺍﻤﻥ ﻟﻌﻘﺎﻗﻴﺭﺃﻴﺩﺭﻭﻜﻠﻭﺭﻴﺩ ﺍﻟﻜﻠﻭﺭﻓﻴﻨﻭﻜﺴﻤﻴﻥ ﻭﺍﻟﻜﺎﻓﻴﻴﻥ ﻭ٨-ﻜﻠﻭﺭﻭﺜﻴﻭﻓﻴﻠﻠﻴﻥ ﺒﺎﺴﺘﺨﺩﺍﻡ ﺠﻬﺎﺯ ﻜﺭﻭﻤﺎﺘﻭﺠﺭﺍﻓﻴﺎ ﺍﻟﺴﺎﺌل ﺫﺍﺕ ﺍﻟﻜﻔﺎﺀﺓ ﺍﻟﻌﺎﻟﻴﺔ ﻋﻠﻰﻋﻤﻭﺩ ﻓﺼل ﻤﻥ ﻨﻭﻉ ﺍﻟﺴﻴﻠﻴﻜﺎ ﻭ ﺒﺎ ﺴﺘﺨﺩﺍ ﻡ ﻤﺯﻴﺞ ﻤﻥ ﺨﻼﺕ ﺍﻹﺜﻴل ﻭﺍﻟﻜﺤﻭل ﺍﻟﻤﻴﺜﻴﻠﻰ ﺒﻨﺴﺏ ﻤﺘﺴﺎﻭﻴﺔ ﻭﻋﻨﺩ ﺃﺱ ﻫﻴﺩﺭﻭﺤﻴﻨﻰ٩ ﻜﻭﺴﻴﻠﺔ ﻤﺘﺤﺭﻜﺔ ﺒﻤﻌﺩل ﺩﻓﻊ ١ ﻤﻠﻴﻤﺘﺭ ﻟﻜل ﺩ ﻗﻴﻘﺔ. ﻫﺫﺍ ﻭﻗﺩ ﺃﻤﻜﻥ ﻗﻴﺎﺱ ﻤﻌﺩل ﺘﺭﻜﻴﺯﺍﺕ ﻤـﻥ ٣٧,٥ - ٠,٧٥،٧٥٠ - ٤٨، ٠,١ - ٤٠ ﻤﻴﻜﺭﻭﺠـﺭﺍﻡ ، ﻟﻜل ﻤﻠﻴﻤﺘﺭ ﻭﺒﻤﻌﺩل ﺇﺴﺘﺭﺠﺎﻉ ﻤﺌﻭﻯ ١٠٠,٠٦ + ٠,٤٧ ، ٩٩,٤٣ + ٠,٦٠ ، ١٠٠,٦٥ + ١,٢٥ ﻟﻜل ﻤﻥ ﺃﻴﺩﺭﻭﻜﻠﻭﺭﻴﺩ ﺍﻟﻜﻠﻭﺭﻓﻴﻨﻭﻜﺴﻤﻴﻥ ﻭﺍﻟﻜﺎﻓﻴﻴﻥ ﻭ٨-ﻜﻠﻭﺭﻭﺜﻴﻭﻓﻴﻠﻠﻴﻥ ﻋﻠﻰﺍﻟﺘﻭﺍﻟﻰ. ﻭﻗﺩ ﻁﺒﻘﺕ ﺍﻟﻁﺭﻴﻘﺔ ﺒﻨﺠﺎﺡ ﻓﻰ ﺘﺤﻠﻴل ﺍﻟﻌﻘﺎﻗﻴﺭﺍﻟﻤﺫﻜﻭﺭﺓ ﻓﻰﺼﻭﺭﺘﻬﺎ ﺍﻟﻨﻘﻴﺔ ﻭﻜﺫﻟﻙ ﻓـﻰ ﺍﻟﻤﺴﺘﺤﻀـﺭ ﺍﻟﺼﻴﺩﻟﻰ ﺍﻟﻤﺤﺘﻭﻯ ﻋﻠﻴﻬﺎ (ﺍﻷﻗﺭﺍﺹ).ﻭﻗﺩ ﺘﻡ ﺍﻟﺘﺄﻜﺩ ﻤﻥ ﺼﻼﺤﻴﺔ ﺍﻟﻁﺭﻴﻘﺔ ﺍﻟﻤﻘﺘﺭﺤﺔ ﺒﺈﺴﺘﺨﺩﺍﻡ ﻁﺭﻴﻘﺔﺍﻹﻀﺎﻓﺔ ﺍﻟﻘﻴﺎﺴﻴﺔ. ﻫﺫﺍ ﻭﻗﺩ ﻗﻭﺭﻨﺕ ﺍﻟﻨﺘﺎﺌﺞ ﺍﻟﺘﻰ ﺍﻤﻜﻥ ﺍﻟﺤﺼﻭل ﻋﻠﻴﻬﺎ ﺍﺤﺼﺎﺌﻴﺎ ﻤﻊ ﺍﻟﻁﺭﻕ ﺍﻟﺩﺴﺘﻭﺭﻴﺔﺍﻟﻤﺨﺘﺎﺭﺓ ﻭﻟـﻡ ﺘﻜﻥ ﻫﻨﺎﻙ ﺍﻴﺔ ﻓﺭﻭﻕ ﺍﺤﺼﺎﺌﻴﺔ.
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