Physicochemical Analysis Semester IV/ IBy Ida Musfiroh

Deskripsi Perkuliahan : Mempelajari Metode metode analisis senyawa dengan instrumentasi yang meliputi : prinsip penggunaan, susunan instrumentasi, fungsi tiap komponen, jenis komponen instrumentasinya, kegunaan dalam analisis kualitatif dan kuantitatif, parameter analisis

Metode instrumentasinya meliputi : Spektrofotometri UV/Vis, Flame Emisi Spektroskopi, Atomic absorption spectroskopi, spektroskopi massa, dan metode metode elektrokimia.

Tujuan Instruksional : Mahasiswa dapat :Membedakan/memilih metode analisis senyawa secara instrumental sesuai dengan sifat fisikokimia senyawa.Menjelaskan parameter ouput dari hasil analisis baik kualitatif maupun kuantitatif.Dapat memilih metode analisis untuk tujuan kualitatif maupun kuantitatif

Manfaat Kuliah :

Mahasiswa dapat Mengaplikasikan metode analisis berdasarkan sifat fisikokimia senyawa dalam sampel obat, makanan ataupun kosmetik dengan metode yang tepat.

Strategi Perkuliahan :

Kuliah dilakukan dengan cara Ceramah di kelas dengan media in focus dalam bentuk slide power point, tanya jawab dan diskusi kelompok.

Buku bacaan pokok dalam perkuliahan ini adalah :

Skoog, D.A. 2003, Principles of Instrumental Analysis, fifth ed, Saunders College Publishing.William, D.H., and Fleming, I. (1980), Spectroscopic Methods in Organic Chemistry third ed, Mcgraw-Hill Book, Company.Christian D.G., 1994, Analytical Chemistry, Canada : John Wiley & SonsDonald L.Pavia, Gary M.Lampman, George S Kriz, 1979, Introduction to Spectroscopy : a guide for Students of Organic Chemistry, Philadelphia : Saunders Golden Sunburst Series, W.B.Saunders Comp. Wang, J., 2000, Analytical Electrochemistry, 2nd edition, New York : Wiley-VCH,

Scoring/Penilaian :

80: A70-79: B60-69: C50-59: D50: E

Pembobotan nilai adalah sebagai berikut :

Nilai Tugas: 15%Kuis: 15%UTS: 35%UAS: 35%

Materi KuliahIntroduction and Physicochemical Properties part IPhysicochemical Properties part II/ExerciseBasic Principles of Instrumental of Analytical Methods Basics and Chemical bonding of Molecular AbsorptionInstrumentation, qualitatif and quantitatif aspects of UV/Vis Spectrophotometry AnalysisPhosphorescens and FluorometryPhosphorescens and Fluorometry/ ExerciseUTS

Atomic absorption spectrometry (AAS)Atomic absorption spectrometry (AAS)ICP Flame emission spectrometry (FES)Electrochemical Methods Electrochmeical MethodsMass Spectrometry ExerciseUAS

PHYSICOCHEMICAL PROPERTIES Semester IV/ II*

An oral drug must be able to:dissolvesurvive a range of pHs (1.5 to 8.0)survive intestinal bacteriacross membranessurvive liver metabolismavoid active transport to bileavoid excretion by kidneyspartition into target organavoid partition into undesired places (e.g. brain, foetus)

What must a drug do other than bind?liverbileductkidneysbladderBBB

Structure - Physiochemical properties

Acid / base propertiesWater solubilityPartition coefficient(Crystal structure)StereochemistryHuman body: ca 75% waterpH blood ca 7.4 (physiology pH)pH stomach 1 - 3.5pH duodenum ca. 4pH urine ca. 6

Identification of acidic / basic functional groupspKa determines degree of ionization different places in the body

*- Recognition of acidic or basic organic functional groups1- Common acidic organic functional groupsCarboxylic acid (-COOH)Phenol (Ar-OH)Sulfonamide (R-SO2NH2)Imide (R-CO-NH-CO-R)-Carbonyl group (-CO-CHR-CO-)

*Recognition of acidic or basic organic functional groups(cont)

2- Common basic organic functional groups

Aliphatic 1 (R-NH2), 2 (R2NH) and 3 (R3N)-amines Heterocyclic aminesAromatic amines (Ar-NH2)

*Estimation of the Relative Acid/Base StrengthThe ionization constant (ka) indicates the relative strength of the acid or base.

An acid with a ka of 1x10-3 is stronger acid (more ionized) than one with a ka of 1x10-5

A base with a ka of 1x10-7 is weaker (less ionized) than one with a ka of 1x10-9

The negative log of the ionization constant (pka) also indicates the relative strength of the acid or base.

An acid with a pka of 5 (ka=1x10-5) is weaker (less ionized) than one with pka of 3

Whereas a base with a pka of 9 is stronger (more ionized) than one with a pka of 7

E.g. Ionization of weak acid (e.g. acetic acid, pka =4.76) is as follows:

*Estimation of the Relative Acid/Base StrengthThe following chart is comparing acid/base strengths:

*The following chart is comparing acid strengths of various functional groupsThe following chart is comparing base strengths of various functional groups

*Ionization of Acidic and Basic Functional GroupsI-AcidsII-Bases

*Acidic and Basic Functional Group - Salt Formation Salt: is the combination of an acid and a base

The salt form of the drug is more soluble than its parent moleculeDrug salts can be divided into two classes: Inorganic salts: are made by combining drug molecules with inorganic acids and bases, such HCl, H2SO4, KOH and NaOH. Inorganic salts are generally used to increase the aqueous solubility of a compound Organic salts: are made by combining two drug molecules, one acidic and one basic. The salt formed by this combination has increased lipid solubility and generally is used to make depot injections (e.g. procaine penicillin).

KJM5230-H06Cyclopentolate - tertiary amine, pKa ca. 10 = active formpH=pKa; [acid]= [base]pHpKa; basic form dominates

KJM5230-H06

KJM5230-H06Antibacterial sulfonamidesOld compoundModern compoundbase form, ionic, water sol.

KJM5230-H06

Henderson-Hasselbach equation: pH = pKa + log ([conjugate base]/[acid]) Example: What is the ratio of pseudoephedrine to pseudoephedrine HCl (pKa = 9.9) in the small intestine at pH = 8.0? 8.0 = 9.9 + log ([pseudoephedrine]/[HCl salt]) log ([pseudoephedrine]/[HCl salt]) = -9.9 + 8.0 = -1.9 ([pseudoephedrine]/[HCl salt]) = 0.0126 13 pseudoephedrine per 1000 pseudoephedrine HCl Ionization vs. pH

% Ionized vs. pHFor HA acids:% ionization = 100/(1 + 10(pKa pH)) For BH+ acids: % ionization = 100/(1 + 10(pH pKa)) Example: Percentage ionized pseudoephedrine HCl (pKa 9.9) in the small intestine at pH 8.0? % ionization = 100/(1 + 10(8.0 9.9)) % ionization = 100/(1 + 0.0126) % ionization = 100/1.0126 % ionization = 98.76%

IonisationIonisation = protonation or deprotonation resulting in charged moleculesAbout 85% of marketed drugs contain functional groups that are ionised to some extent at physiological pH (pH 1.5 8).The acidity or basicity of a compound plays a major role in controlling:

Absorption and transport to site of action Solubility, bioavailability, absorption and cell penetration, plasma binding, volume of distributionBinding of a compound at its site of action un-ionised form involved in hydrogen bondingionised form influences strength of salt bridges or H-bondsElimination of compoundBiliary and renal excretionCYP P450 metabolism

So the same compound will be ionised to different extents in different parts of the body.

How does pH vary in the body?

FluidpHAqueous humour7.2Blood7.4Colon5-8Duodenum (fasting)4.4-6.6Duodenum (fed)5.2-6.2Saliva6.4Small intestine6.5Stomach (fasting)1.4-2.1Stomach (fed)3-7Sweat5.4Urine5.5-7.0

pKa = 4.1Ionisation of an acid 2,4-dinitrophenol

pKa = 9.1Ionisation of an base 4-aminopyridine

Chart2

0.000079432899.9999205672

0.000125892499.9998741076

0.000199525899.9998004742

0.000316226899.9996837732

0.000501184799.9994988153

0.000794321999.9992056781

0.001258909699.9987410904

0.001995222599.9980047775

0.003162177799.9968378223

0.005011621299.9949883788

0.007942651499.9920573486

0.012587669499.9874123306

0.019948642999.9800513571

0.031612779899.9683872202

0.050093617199.9499063829

0.079369777899.9206302222

0.125734251199.8742657489

0.199128917199.8008710829

0.315230918399.6847690817

0.498687873799.5013121263

0.78806838599.211931615

1.243273525498.7567264746

1.956230387398.0437696127

3.065343003296.9346569968

4.772672103495.2273278966

7.358755611892.6412443882

11.181576977888.8184230222

16.633753081783.3662469183

24.025307335275.9746926648

33.386057541766.6139424583

44.268836623855.7311633762

55.731163376244.2688366238

66.613942458333.3860575417

75.974692664824.0253073352

83.366246918316.6337530817

88.818423022211.1815769778

92.64124438827.3587556118

95.22732789664.7726721034

96.93465699683.0653430032

98.04376961271.9562303873

98.75672647461.2432735254

% neutral

% cation

pH

percent

monoacid

pH% neutral% anionpKa

394.06490568975.93509431034.2

3.290.90909090919.0909090909

3.486.319311139713.6806888603

3.679.923999108720.0760008913

3.871.525275104928.4747248951

461.313682015338.6863179847

4.25050

4.438.686317984761.3136820153

4.628.474724895171.5252751049

4.820.076000891379.9239991087

513.680688860386.3193111397

5.29.090909090990.9090909091

5.45.935094310394.0649056897

5.63.828650388296.1713496118

5.82.45033675597.549663245

61.560166224298.4398337758

6.20.990099009999.0099009901

6.40.627001234199.3729987659

6.60.396528561999.6034714381

6.80.250559266799.7494407333

70.158238528199.8417614719

7.20.099900099999.9000999001

7.40.063055948899.9369440512

7.60.039794874499.9602051256

7.80.025112556399.9748874437

80.015846420499.9841535796

8.20.009999000199.9900009999

8.40.006309175499.9936908246

8.60.003980913299.9960190868

8.80.002511823399.9974881767

90.001584868199.9984151319

9.20.0009999999.99900001

9.40.000630953499.9993690466

9.60.000398105699.9996018944

9.80.00025118899.999748812

100.000158489199.9998415109

10.20.000099999999.9999000001

10.40.000063095799.9999369043

10.60.000039810799.9999601893

10.80.000025118999.9999748811

110.000015848999.9999841511

&A

Page &P

monoacid

&A

Page &P

% neutral

% anion

pH

percent

monobase

7% neutral% cationpKa

30.000079432899.99992056729.1

3.20.000125892499.9998741076

3.40.000199525899.9998004742

3.60.000316226899.9996837732

3.80.000501184799.9994988153

40.000794321999.9992056781

4.20.001258909699.9987410904

4.40.001995222599.9980047775

4.60.003162177799.9968378223

4.80.005011621299.9949883788

50.007942651499.9920573486

5.20.012587669499.9874123306

5.40.019948642999.9800513571

5.60.031612779899.9683872202

5.80.050093617199.9499063829

60.079369777899.9206302222

6.20.125734251199.8742657489

6.40.199128917199.8008710829

6.60.315230918399.6847690817

6.80.498687873799.5013121263

70.78806838599.211931615

7.21.243273525498.7567264746

7.41.956230387398.0437696127

7.63.065343003296.9346569968

7.84.772672103495.2273278966

87.358755611892.6412443882

8.211.181576977888.8184230222

8.416.633753081783.3662469183

8.624.025307335275.9746926648

8.833.386057541766.6139424583

944.268836623855.7311633762

9.255.731163376244.2688366238

9.466.613942458333.3860575417

9.675.974692664824.0253073352

9.883.366246918316.6337530817

1088.818423022211.1815769778

10.292.64124438827.3587556118

10.495.22732789664.7726721034

10.696.93465699683.0653430032

10.898.04376961271.9562303873

1198.75672647461.2432735254

&A

Page &P

monobase

&A

Page &P

% neutral

% cation

pH

percent

amphoteric

pH% Neutral% Cation% Anion% ZwitterionpK(C to Z)pK(C to N)pK(Z to A)pK(N to A)

40.000399.99940.00000.00039.549.5810.0810.041.109.2610.36

4.20.000499.99910.00000.00053

4.40.000799.99860.00000.0007I2 to L2 are calculated automatically

4.60.001099.99780.00000.0012

4.80.001799.99650.00000.0018

50.002699.99450.00000.0029

5.20.004299.99120.00000.0046

5.40.006699.98610.00000.0073

5.60.010599.97800.00000.0115

5.80.016699.96510.00000.0183

60.026399.94480.00000.0289

6.20.041799.91250.00000.0458

6.40.066099.86140.00000.0726

6.60.104599.78050.00000.1150

6.80.165499.65250.00010.1820

70.261699.45030.00020.2879

7.20.413299.13130.00060.4548

7.40.651698.62970.00150.7172

7.61.024697.84410.00371.1276

7.81.603596.62230.00931.7649

82.492094.74240.02292.7427

8.23.830991.89700.05574.2164

8.45.794087.69560.13356.3770

8.68.556581.71370.31249.4174

8.812.218973.62590.706913.4483

916.682863.42611.529718.3614

9.221.529551.64583.128823.6958

9.426.011139.36945.991128.6284

9.629.234127.918410.671832.1757

9.830.471718.361017.629633.5377

1029.431111.189426.987032.3925

10.226.35906.323138.306829.0112

10.421.95263.322750.563224.1615

10.617.10401.633462.437518.8250

10.812.58420.758372.807113.8504

118.84320.336281.08779.7330

11.26.00340.144087.24526.6074

11.43.97640.060291.58704.3765

11.62.58970.024794.53532.8503

11.81.66770.010096.48671.8355

121.06610.004197.75651.1734

&A

Page &P

amphoteric

&A

Page &P

% Neutral

% Cation

% Anion

% Zwitterion

pH

percent

Sheet5

&A

Page &P

Sheet6

&A

Page &P

Sheet7

&A

Page &P

Sheet8

&A

Page &P

Sheet9

&A

Page &P

Sheet10

&A

Page &P

Sheet11

&A

Page &P

Sheet12

&A

Page &P

Sheet13

&A

Page &P

Sheet14

&A

Page &P

Sheet15

&A

Page &P

Sheet16

&A

Page &P

&A

Page &P

Structure - Physiochemical propertiesAcid / base propertiesWater solubilityPartition coefficient(Crystal structure)StereochemistryIonisation -permanent charge -acid / base properties

Hydrogen bonds

The more H-bonds possible - the more water sol.

*The presence of oxygen and nitrogen containing functional groups usually enhances water solubility. While lipid solubility is enhanced by nonionizable hydrocarbon chains and ring systems.

Solubility Prediction

KJM5230-H06logPExperimental: MlogP or logPmeasP: Partition coefficient between n-octanol and waterClogP (SciFinder): 2.69p-value: hydrophilic - lipophilic valuelogP Rt (HPLC, TLC reverse phease)Structure - Physiochemical propertiesAcid / base propertiesWater solubilityPartition coefficient(Crystal structure)StereochemistryCalcd: ClogP

KJM5230-H06

Fragment

-value

C (aliphatic

+0.5

Phenyl

+2.0

-Cl

+0.5

-ONO2

+0.2

-S-

0.0

O=C-O- (carboxyl)

-0.7

O=C-N- (amide)

-0.7

-O- (hydroxyl, ether)

-1.0

N (amine)

-1.0

-NO2 (aliphat.)

-0.85

-NO2 (aromat.)

-0.28

KJM5230-H06Absorption of Bioactive CompoundsAbsorption from GI tract

KJM5230-H06

Most drugs: Passive diffusionLow lipophilicity unionized form - low absorption

logP - P: Partition coefficient between n-octanol and water

If a compound can ionise then the observed partitioning between water and octanol will be pH dependent.Distribution coefficients

Relationship between logD, logP and pH for an acidic drug Indomethacin

pH - Distribution behaviour of bases

pH - Distribution behaviour of amphoteric compounds

pH Effects on Absorption

Drug Transfer Acidic Drugs

Drug Transfer Acidic Drugs

Drug Transfer Basic Drugs

Drug Transfer Basic Drugs

Contoh SoalSoal : Jika asam asetat (pKa=4,76) dalam larutan pH 4,76. Hitung derajat ionisasinya.

Penyelesaian

Menurut persamaan Handerson-Hasselbalch

CH3COOH ------ CH3COO- + H+

pH = 4,76 + log (CH3COO-)/(CH3COOH)

Maka kita dapat menentukan derajat ionisasi asam asetat pada pH tersebut, yaitu :4,76 = 4,76 + log (CH3COO-)/(CH3COOH)log (CH3COO-)/(CH3COOH) = 0(CH3COO-)/(CH3COOH) = 100 = 1Derajat inosisasi = 1Artinya asam asetat akan mengalami ionisasi 50% pada pH 4,76, atau jumlah asam asetat yang terionisasi sama dengan jumlah asam asetat yang tidak terionisasi.

Latihan soal 1Soal : Jika Amonia (pKa = 9,25) dalam larutan pH 9,25, tentuka derajat ionisasinya.

Penyelesaian Menurut persamaan Handerson-Hasselbalch :NH4+ ---- NH3 + H+ pH = pKa + log (NH3)/(H+)9,25 = 9,25 + log (NH3)/(H+)log (NH3)/(H+) = 0 (NH3)/(H+) = 100 = 1Derajat ionisasinya = 1

Latihan soal 2Tentukan derajat ionisasi asam asetat (pKa=4,76) pada pH (i) 3,76 (ii) 5,76

Latihan soal 3 Hitung persentase ionisasi amonia pKa = 9,25 pada

(i) pH : 8,25(ii) pH : 10,25

Latihan soal 4Hitung persentase ionisasi obat pada pH 7 : Difenhidramin pKa = 9Ibuprofen pKa= 4,4

Profil sifat fisikokimiaProkain

Gugus fungsional :A : amin alifatis tersierB : Ester netralC : Amin aromatis primer

Merup senyawa Basa sangat lemah pKa 2

t1/2 dlm air pd pH 7, 37C = 26 hari

ProkainObat anastesi lokal

Molekul obat yang berhubungan erat dengan prokain : benzokain, ametokain, butakain, propoksikain, prokainamid, buvikain

Reaksi hidrolisis Prokain :

Profil sifat fisikokimiaParasetamol

Gugus fungsional :

A. gugus amida, netralB. Gugus hidroksi fenolik, asam lemah pKa = 9,5

Merupakan obat analgetika-antipiretik

Latihan soal 5

Jika telah mengetahui sifat fisikokimia berdasarkan gugus fungsi dari dua senyawa tersebut, maka metode apakah yang dapat digunakan untuk menganalisis kadar dari dua senyawa tersebut?

*A drug needs to be able to do many things other than bind to the active site of the receptor! This diagram illustrates a model for an oral drug to treat a problem with the foot. It is a very complicated model of a human, and its hard, if not impossible, to measure all these processes directly. In addition, it is much better to have an in vitro model than have to carry out these tests in living animals or whole organs.

BBB = blood brain barrier

********Drugs are only absorbed passively when they are unionised. This is because the compound has to pass through a lipophilic (fat loving) membrane and this process will be unfavourable for charged molecules. In a more acidic medium, such as the stomach, the percentage ionised for an acidic compound will be less than at pH 7.4 and so more compound will have the capacity to be passively absorbed. In comparison, a basic compound in an acidic medium will be more ionised and so less of the compound will be in the neutral form and have the capacity to undergo passive absorption. This may well be the reason for the observation that acidic compounds generally have better fraction absorbed (hence bioavailability) than bases with neutral compounds lying between them.

*The extent of ionisation of a compound can have a large effect on many biological properties, such as receptor/enzyme binding, binding to plasma proteins, CNS penetration, solubility and absorption. Once the pKa value of a molecule is known, then it is possible to calculate the proportion of ionised and neutral species at any pH. *This diagram shows the proportions of phenol and phenolate anion at pHs between 3 and 11. Above about pH 6 the phenol is completely deprotonated. When the pH is the same as the pKa (i.e. 4.1 in this case), half the molecules are ionised and half are not. Note that phenols are significantly more acidic than aliphatic alcohols, and that 2- and 4-nitrophenols are more acidic than phenol itself. Do you know the pKa of, for example, ethanol?*And here is the corresponding diagram for a basic compound. Below about pH 7, the molecule will be completely protonated. Do you know why the ring nitrogen protonates rather than the NH2?**Lipophilicity may be modelled using simple physical chemical models. The partition coefficient P is a measure of lipophilicity and is usually experimentally determined by equilibrating a sample of the compound in an octanol/aqueous buffer mixture. The resulting emulsion is then separated. Once separated, the concentration of the drug in each layer is measured and the partition coefficient is then calculated.*Ionisation of a compound (either an acid or a base) will favour the distribution of the drug into the aqueous phase since the concentration of the neutral form of the compound, which is the only form of the compound capable of partitioning into the octanol phase, is reduced. *If we can calculate a theoretical pKa and logP, these equations allow the calculation of a theoretical logD.*An amphoteric compound can act as either an acid or a base.*mq-fig-01-05-0.jpg