application of HPLC & GC


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Seminar on application of HPLc and Gc:

Prepared By: Jigar Goswami M.Pharm 1 st Semester Roll No.12 Guided By: Dr.Nehal J.Shah M.Pharm, Ph.D. Principal Of DDPC Dept. of P’ceu Chemestry Dharmaj Degree Pharmacy College Dharmaj Seminar on application of HPLc and Gc 1

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2 Application of HPLC: There are mainly two types of application: Qualitative Analysis Quantitative Analysis

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QUALITATIVE ANALYSIS HPLC is used for identification of compound: Here comparison of retention time of test sample with the reference compound carried out. Checking purity of compound : Purity of compound checked by comparison of chromatogram of test with the reference standard. Presence of impurities: If impurities present into sample we observed additional peak when we compared the chromatogram of test with the reference standard. 3

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QUANTITATIVE ANALYSIS HPLC is used for assay of many drugs like cephalosporin, furosemide. It is also used into drug mixture determination. Biopharmaceutical and pharmacokinetic study. S tability study Purification of some compound of natural or synthetic origin. Investigation of biological material such as gastric content, blood, urine sample etc. are done by HPLC. Many poisonous substances can also be investigated by HPLC. INGORGANIC CHEMISTRY Chromatographic separation of anion like I-,IO-, ClO-4 can be done effectively by using ion exchange chromatography 4

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BIOCHEMICAL ANALYSIS LIPID Separation of reference mixtures of glycerides, fatty acid can be done on silica column. AMINO ACID HPLC is widely used for analysis and separation of amino acid and protein. For this size exclusion chromatography is used. CARBOHYDRATE HPLC is suitable for analysis of carbohydrates. HPLC is used for determination of sugar content of soya bean extracts, daily products. 5

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NUCLEIC ACID HPLC is used into all area of nucleic acid research. HPLC is used for analysis of nucleic acid, separation of starting material, intermediates, separation and purification of nucleic acid. VITAMINS HPLC is used for analysis of vitamins in variety of food products and animal feeds. 6

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ISOLATION OF NATURAL PHARMACEUTICAL ACTIVE COMPOUNDS Some plant containing alkaloid and glycosides. From that alkaloid and glycosides can be isolated by means of HPLC. PETROCHEMICAL INDUSTRY HPLC is used for rapid separation of petroleum products and coal products with a wide boiling range using silica gel as stationary phase. ENVIRONMENTAL POLLUANTS HPLC is used for detection of level pesticides and carcinogens in air, water, food supplies. Organophosphate like Parathion, Melathion can be analysed by HPLC. 7

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8 Application of G.C. Qualitative Quantitative Qualitative It is done by comparing Rt and Rv of sample component with Rt and Rv of reference compound developed under identical experimental condition. It is done either by measurement of Rt of sample component & comparing with Rt of separately injected known compound or recording chromatogram of sample and then recording chromatogram of sample as well as known compound. If spiked sample shows and increase in area under curve of its peak it is due to added compound.

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9 Analyst should use 2 or more column packed with stationary phase of different polarity for qualitative analysis of sample. If analysis is to be performed with Rt / Vol of known compound developed on different days and on different experimental conditions, then usually usually relative retention or specific retention volume of Kovat’s retention indices are compared. The Kovat’s retention index I relates the retention of sample component of straight chain H.C. which elute before and after sample component.

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10 I = 100 [log (t – tm) – log ( tn – tm) / log (t n+1 – tm) – log ( tn - tm )] + 100n =100 [log (v – vm ) – log ( vn – vm ) / log (vn+1 – tm) – log ( vn - vm ) + 100n I = 100 [log αs , n / log αn+1,n] +100 n Where t= retention time of sample tm =retention time of mobile phase tn = retention time of saturated hydrocarbon containing n carbon which elute before sample tn+1= retention time of saturated H.C. containing n+1 carbon which elute just after sample.

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11 A. Peak area determination 1. Mechanic or electronic integration modern electronic integrators and computers can determine peak areas with standard deviations of 0.5% or less. 2. Triangulation. It involves constructing the triangle formed by drawing tangents to the peak inflections points and calculating the area of this triangle. This is most popular manual method but its applicability to asymmetric peak is poor.

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12 3.Planimetry - Planimeter is a device which determines area by tracing around the perimeter of figure. It is slow and tedious method 4.Cut and weigh method -The original peaks are cut and weighed on an analytical balance. Slow and imprecise method 5.Retention time method -This method involves arbitrary area determinations by multiplying peak height times retention time.

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13 B. Peak height determination Some electronic integrators and computers can determine peak height as well as area. Since peak heights are quite dependent on the chromatographic conditions the internal standard technique is highly recommended for optimum precision. C. Data interpretation 1.Area Normalization Here concentration of component in a mixture is defined as the percentage of the total peak area represented by the individual component peak area.

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14 Ci % = Ai / Σ n Ai * 100 Where, Ci = % composition of the I’th component. Ai= chromatographic peak area of the ith component ΣAi = summation of areas of all component peaks in the chromatogram.

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15 Maximum accuracy is obtained when all the component of a mixture are structurally similar. This method is frequently used in HC mixture analysis. Advantage One does not need calibration standards for this type of quantitation . Disadvantage Equal detector response is assumed for all components and this may not be valid for mixtures of components with widely varying structures.

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16 2. Area normalization with Response factor correction Area of a component peak is proportional to its concentration. Ai = fi Ci Where, Ai = area of the it peak Ci = concentration of ith component Fi = proportionality factor for ith component Ci % = fi Ci / Σfi Ai *100 Where, Ci % = concentration of ith component in %. Fi = response factor for the ith component Ai = area of ith component

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17 Response factor may be calculated as follows Fi = component amount / peak area It is determined by measuring the peak area for a known quantity of pure material Disadvantage Pure substance are required as standards. However if pure substance are not available, then relative molar response from similar compound are determined.

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18 3.External standardization Here the standard is separate than the sample. This procedure involves calibration of the instrument by injection of samples of known quantitative composition. The calibration is performed via plot of peak area vs. concentration. Disadvantage Accuracy and precision may be less because success of technique is dependent on highly reproducible injection volume. So internal standardization is preferred.

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19 4. Internal standardization This method involves the addition of a compound the internal standard, which is not already present in the sample. Usual guidelines for selection of a compound as internal standards are:- It should be structurally similar to the compound to be analyzed. The internal standard should be separated from, but elute close to the sample compound. The internal standard peak should be similar to the sample peak in size.

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20 A1 = f1C1 ……..for sample A2 = f2 C2 …….for internal standard Taking ratio A1/A2 = f1C1/ f2C2 A1/A2= K C1/C2 Where K = constant determined by f1/f2. Thus a plot of A1/A2 vs C1/C2 should be a straight line with zero intercept and slope of f1/f2. The factor A1/A2 is called peak area ratio. While C1/C2 = wt ratio.

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21 The amount of internal standard is same as that of sample. So C2 is constant which can be incorporated in slope constant so we can have A1/A2 = K C1 Where K = f1/f2 C2. Thus a plot of peak area ratio vs amount of concentration of sample should be straight line which is used as calibration curve for the assay. Advantage It eliminates the dependence of quantitation on injection volume because the peak area or height ratio is dependent only on weight ratio and not on amount injected.


References: Douglas A. skoog, F James Holler, Timothy A. Nieman, ‟Principle of instrumental analysis”, Thomson Asia Pvt. Ltd., Singapore, 2004, Fifty Edition, P. -411 to 426. Sharma B.K., ‟Instrumental method of chemical analysis”, Krishna prakashan Media, U.P., 2005, Twenty forth edition, P.-S-313 to s-315. Chatwal G.R., Anand S.K., Instrumental methods of chemical analysis, Himalaya Publishing House, Mumbai, 5 th Edition, Page No.2.566-2.587. 4. Kasture A.V., Mahadik K.R., Pharmaceutical Analysis Nirali Prakashan, Volume 2, Page No. 4-47. 22

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