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PRAKTIKUM I KINETIKA ENZIM Pengaruh pH Terhadap Aktivitas Enzim Reagen :

Larutan Enzym (pankreatin atau amilase?) 1 (w/v) 1 gram enzim Aquadest ad 1000 ml Ambil labu ukur 1 L, timbang enzim 1 g masukkan labu ukur, lalu tambah aquadest sampai 500 ml, campur (mix), tambah aquadest sampai dekat garis, mix, lalu tetesi dengan aqadest sampai garis 1 L

NaCl 0.9 % (w/v) Larutan Substrat (amylum 1%)

0,9 gram NaCl, aquadest ad 100 ml 1 gram amilum, aquadest ad 100 ml (bukan 125 ml)

Penyangga pH: 4; 5; 6.5; 8; 103333 KI - KIO3 : 1. KI 5 gram

2. KIO3 0.375 gram 3. NaOH 1 M 2 ml


HCl 0.05 M HgCl 2% (w/v)

aquadest netral ad 1 liter

4,15 ml add 1 liter aquadest (HCL 37%) 2 gram HgCl2, aqua ad 100 ml

Pembuatan; 1. pH 4 & pH 5

Acetate Buffer (sodium acetate-acetic acid buffer) pH 4-5.6 Sodium acetate 0.2M = 27.2 gm/1 (MW - 136.09)

CH3COONa.3H2O Acetic acid CH3COOH 0.2M

(MW = 60)

Add sodium acetate to acetic acid to give desired pH. Dilute with ddH20 (aquabidestilata = double distilled water) to desired molarity

Di Lab biokimia Na Acetat 0.2 M 0.2 x BM CH3COONa ( 82.04) = 16,408 gram, aquadest ad 1L 32,816 g / 2liter Kalau BM 136,09 (CH3COONa.3H2O ) maka Na asetat 27,2 g Asam Asetat 0,2 M BM = 60,05 ; 1 liter = 1,05 kg = 60 x 0,2 x 1000 = 11,438 ml 1050 g pH 4 = 17 ml Na Asetat + 83 ml As.Asetat 0,2 M atau = 170 bag Na Asetat + 830 bag As.Asetat 1 liter Aturlah dengan penambahan sedikit asam asetat atau Na asetat kalau pH kurang pas pH 5 = 68 ml Na asetat + 32 ml asam asetat 0,2 M atau di lab biokimia 7 bag Na Asetat + 3 bag As.Asetat = 700 bag Na Asetat + 300 bag As.Asetat 1 liter 2. pH 8 & 10 Borate Buffer pH 7.4-9.2 0.2M = 76.2 gm/ml (MW = 381.37)

Borax (sodium tetraborate) Na2B407.120H20 Boric acid H3BO>3

0.2M = 12.37 gm/1 (MW = 61.83)

Add boric acid to borax solution until desired pH is reached. Dilute to desired molarity with ddH20 Di Lab. Biokimia agak beda Hitungan? Reagen A : 12,4048 g as.borat (boric acid) + 14,912 g KCl 1 liter Reagen B : NaOH 0,2 N 8,002 g, aquadest ad 1 liter

pH 8 = 500ml A + 3,97 ml B, aqua ad 200 ml = 250 A + 19,85 B 1 liter pH 10 = 50 ml A+ 43,9 ml B add aqua 200 ml = 250 A + 219 ml B 1 liter

3. pH 6,8
Plummer: Buffer Na fosfat: x ml 0,2 M NaOH dimasukkan ke 50 ml 0,2 M Na H2PO4 Buffer K fosfat: x ml 0,2 M KOH dimasukkan ke 50 ml 0,2 M KH2PO4 x ml pH x ml pH 3,5 5,8 43 7,6 5,8 6,0 45 7,8 9,1 6,2 47 8,0 13 6,4 18 6,6 24 6,8 30 7,0 35 7,2 40 7,4

Lalu encerkan (tambah aquadest) sampai garis 100 ml buffer 0,2M Di Lab. Biokimia: Buffer 0,1M Reagen A berisi KH2PO4 0,2 M 27,232 g, aqua ad 1 liter (Lihat dulu BM KH2PO4 pada botol) NaOH 0,2 M pH 6,8 = 50 ml A + 23,65 ml NaOH, aqua ad 200 ml (berarti buffer 0,1M) = 250 ml A + 118,25 ml NaOH, aquadest add 1 liter Atau pH 6,5 = 6 gr KH2PO4 + 2 gr Na2HPO4 cat:atan: - Siapkan 30 buah rak@5 tabung, 1 vol pipet 1 ml , erlenmeyer - Cek pH tiap penyangga - Siapkan buret @ reagen + beaker glass 1 liter (Hitungan?)

- Siapkan cuvet & nyalakan spektrofotometnya - Tiap akan praktek, lakukan percobaan terlebih dahulu sehari sebelumnya Standardization buffers pH=7.00 : Add 29.1 ml of 0.1 molar NaOH to 50 ml 0.1 molar potassium dihydrogen phosphate. Alternatively :

Dissolve 1.20g of sodium dihydrogen phosphate and 0.885g of disidium hydrogen phosphate in 1 liter volume distilled water.

Make up the following solutions (1) 0.1M disodium hydrogen phosphate (14.2g / l) (2) 0.1M HCl (3) 0.1M NaOH Mix in the following proportions to get the required ph ph vol. of phosphate vol. of 0.1M HCl vol. of 0.1M NaOH 7 756.0 mls 244 mls 8 955.1 mls 44.9 mls 9 955.0 mls 45.0 mls 10 966.4 mls 33.6 11 965.3 mls 34.7
Working buffer: 0.1M 100 ml Mix X ml of 0.2M dibasic sodium phosphate with Y ml monobasic sodium phosphate. Dilute to 100 ml with ddH20 pH (25 C) X ml Y ml

5.8 6.0 6.2 6.4 6.6 6.8 7.0 7.2 7.4 7.6

4.0 6.15 9.25 13.25 18.75 24.5 30.5 36.0 40.5 43.5

46.0 43.75 40.75 36.75 31.25 25.5 19.5 14.0 9.5 6.5

7.8 8.0

45.75 47.35

4.25 2.65

PRAKTIKUM II PENGARUH SUHU PADA RX. ENZIMATIK

Reagen yang digunakan sama seperti Praktikum I, tapi hanya Siapkan es batu, penangas air

menggunakan buffer pH 6,5

PRAKTIKUM III PENGARUH AKTIVATOR, INHIBITOR, DAN KADAR ENZIM

Reagen yang digunakan sama seperti Praktikum I HgCl2 1% Mengggunakan buffer pH 6,5 PRAKTIKUM IV BEBERAPA CONTOH ENZIM

A.

UREASE Ureum 1 % (w/v) (buat 3x) PP 1 % usahakan baru

- 1 gr PP, alkohol 95% ad 100 ml


PP 2% (w/v) Urease

- Bila pakai kedelai, 2% dilarutkan dalam NaCl 0,9 %


HgCl2 2%

B.

SUKSINAT DEHIDROGENASE (KATAK) Larutan penyangga phospat pH 6,8 Natrium Suksinat 0,05 N Cara buat: 1 gral = 2 grek ( 1 mmol = 1 meq) BM = 198,13 0,05 x BM = 4,95325 gr, aquadest ad 1 liter 2

Methylene Blue ( 1: 20.000) = w/v PARAFIN

0.05 gr / 1000 ml

C.

Daging ayam dicincang dimasukkan 3 tabung, @ 1 gram

Enzim Schardinger bahan susu (baru) 25 mg methylene blue dilarutkan dlm 195 ml air dan 5 ml formaldehid 40%

Reagen Methylene Blue Formaldehide

PARAFIN

D.

PEROXIDASE SUSU BENZIDIN 4% (w/v) DAN 1 ml H2O2 3% 4,33 ml as.cuka glasial dimasukkan erlenmeyer + ( 0,5 gr Benzidin (dibuat baru) + aquadest kira-kira 20ml) + dipanasi 50o C apabila diencerkan sehingga volume 100 ml (pindah ke labu ukur 100 ml, bilas erlenmeyer dengan aquadest , masukkan labu ukur tersebut, sampai volume larutan 100 ml, berarti kadarnya menjadi 4%

H2O2 3%

(w/w)

Lihat botol kadar berapa? (w/w = 30%?) , lalu encerkan (10X?) Cat:

Siapkan 3 Kertas saring

penangas air 37o C, 2 mendidih simpan dalam refrigerator

BENZIDIN 4% DAN H2O2 3% Susu harus baru

PRAKTIKUM VII CAIRAN EMPEDU, EMPEDU, VITAMIN & INDOL --belum

R. Pettenkofer

R. Hay -

H2SO4 pekat empedu encer (10x) sukrosa 10% (baru) [2 ml] [ 1 tts] cincin ungu

Aquadest Empedu encer Bubuk belerang mengendap

R. Gmellin

Empedu pekat HNO3 pekat

R. Joles Urina Thymol 5% dlm alkohol FeCl3 0,3 % dlm HCl 37% [5 ml ] Chloroform [ 1 ml ] Vitamin A (tabung harus kering) [5 ml] [ 15 tts]

1. -

Minyak ikan

[ 1 tts ] kulkas

Chloroform kering [ 5-6 tts ] chloroform + Na sulfatanhidrid As. Cuka anhydrida [ 1-2 tts ]

Antimonium Trichlorida (SbCl3) dlm chloroform kering yg baru & jenuh [ 20 tts ]

2. Vitamin B

B1 Alkohol 80 % NaOH 40% Lar. K3Fe(CN)6) Alkohol pekat

[ 1ml ] [ 1 ml ] [ 0,6 ml ] [ 3 tts ] [ 5 ml ]

Bolak balik beberapa kali spy homogen, lihat hasilnya di bawa UV (biru)

3. Vit B2 Alkohol 80% Susu sapi [ 5 ml ] [ 2 ml ]

Homogenkan lalu dilihat di bawah sinar UV (hijau) 4. Vit C (titrasi dg Dip) TCA 10% DIP (suasana basa biru, asam merah muda) 130 mg 2,6 DIP + 500 ml air mendidih lalu setelah dingin + 150 mg NaHCO3, simpan dalam gelap Digunakan vit. C 50 mg dalam 1 liter aquadest Lakukan titrasi, 5 ml vit C + 2 ml TCA 10 % lalu titrasi dengan DIP Perhitungan : 5 1000 1 ml DIP ~ 0.25 A titrasi Ket: x 50 mg = 0.25mg ~ a titrasi

Pembuatan Dip;

untuk B1& B2 2 ml susu + 5 ml alkohol 80% kocok sentrifuge B1 suspensi + 0.6 ml NaOH 40 % + 3 tetes K3Fe (CN)6 2% B2 suspensi dilihat di sinar UV Siapkan sinar UV Sentrifuge Tutup tabung

saring bagi 2

PRAKTIKUM VI PENCERNAAN MAKANAN (SALIVA)

PP 1 Litmus 2 Merah Congo CCl4 NaOH 10% CuSO4 1%

pelarut alkohol 95% pelarut aquadest 0,5 gr merah congo dlm 90 ml air + 10 ml alkohol 95%

As. Cuka (as. Asetat) 5% HNO3 5% AgNO3 1% HCl 2% & 5% BaCl2 2% HgCl2 2% H2SO4 5% KI 2% Amylum 1% FeCl3 2% HNO3 pekat Am. Molibdate 2% Am. Oxalat jenuh Lugol dibuat baru

5 gr iodium/ iodine/iodida ( I2 ) 10 gr KI

aquadest 100 ml

Fehling A

Cat:

34,65 gr CuSo4 dlm 500ml aquadest

Fehling B 125 gr KOH + KNa Tartrat ad 500 ml aquadest

- Siapkan 30 buah rak@17 tabung, erlenmeyer, corong, kapas gulung, kasa - Meletakkan reagen2 pada meja + @1 pipet pasteur - Amilum & aquadest dituang di buret - Memanaskan air untuk px. phospat

PRAKTIKUM V MAKANAN ( AIR SUSU SAPI)

MM / MR 2% baru PP 1% MP / phenol red 1 % Chloroform NaOH 10% 300 ml As. Cuka 6% 500 ml Aceton ( panaskan dalam air mendidih) Fehling A & B Biuret 2% dalam alkohol 50%

I. II. III.
IV. add 1 l R.Millon

3 gr CuSO4 5H2O (dilarutkan dulu dlm 500ml aquadest) 9 gr KNa Tartrat 4H2O 24 gr NaOH (dilarutkan dulu dlm 100 ml aquadest) 5 gr KI ditambahkan terakhir supaya tidak tereduksi

Hg : HNO3 pekat = 1 : 2 HNO3 = 150 ml Hg = 70 gram

Hopkins Cole buat 200 ml

40 gr Mg powder (Na amalgam) + as. Oxalat jenuh dibiarkan semua gas keluar disaring diencerkan 2-3x ( dilakukan di kamar asam & bawahx dikasih es)

HNO3 pekat NaOH 40% Am. Molibdate 2%

Cat: Siapkan cawan porselin Pengukur berat jenis Kertas saring Corong Penangas air Xantoprotein HNO3 pekat + NaOH 40% PO4 Am. Molibdate + HNO3 pekat Pipet pasteur

PRAKTIKUM VIII TITRASI FORMOL

NaOH 0,1 N ditritasi dg As. Oxalat 0,1 N dg PP PP 1 Formalin ( pengenceran 3x) Gelatin 4%

Masukkan gelatin sedikit demi sedikit ke aquadest (air panas) Dibuat pH 8 dg menambah NaOH 40% tetes demi tetes

Pancreatin 4%

Cat: 30 kelompok buat gelatin 1 l 42 gr, pancreatin 200 ml 8 gram AAM 20 kelompok, gelatin 500 ml 20 gr, pancreatin 150 ml 6 Waterbath 37o C

gram

PEMBEKUAN DARAH

K. oxalat 10% Na. citrat jenuh Na. fluorida 10% CaCl2 2% CaCl2 bubuk Prosedur: A. Pencegahan Pembekuan Darah Pembekuan darah dapat dicegah dg mengikat ion C++ dg berbagai reagen. Sediakan 4 tabung ( A, B, C, D ) dan masukkan:

A. Larutan K. Oxalat 10% 2 tetes B. Larutan Na. Citrat jenuh 1 tetes C. Larutan Na. fluorida 10% 2 tetes D. Tidak diberi apa2

Putar2 tabung A,B,C supaya dindingnya basah + @ tabung 5 tetes darah Homogenkan diamkan 5 menit amati ada bekuan/tidak. Untuk membuktikan bahwa pembekuan dicegah karena ion C++ terikat, maka + CaCl2 bubuk pada tabung yang tidak menunjukkan pembekuan terjadi pembekuan B. Pembekuan Darah Tabung A + 2 tetes serum Tabung B + 2 tetes lar. CaCl2 2% Tabung C tidak diberi apa-apa Dalam 3 tabung reaksi A, B, C + @ 1 ml darah oxalat, lalu:

Inkubasi 3 tabung pada 37oC, 10 menit goyang untuk mengetahui pada tabung mana terjadi pembekuan. Hanya pada tabung Ctidak terjadi pembekuan.

Tabung A terjadi pembekuan karena serum mengandung trhombin yang dapat merubah fibrinogen menjadi fibrin tanpa memerlukan C++ Tabung B terjadi bekuan darah karena adanya ion2 Ca yang memungkinkan terbentuknya trombin dari protrombin oleh trombokinase.

GLUKOSA DARAH Stock glucose standard 1000mg - 10 gram dextrose a.p. anhydour, larutkan dalambenzene acid pekat, add 1 liter TCA 5% 5 ml TCA dalam 100 ml aquadest

Orto toluidin 8,6 % buat baru 8,6 ml Orto toluidin + add 100ml asam aetic glasial Simpan dalam botol coklat dalam refrigerator

Procedure:

TCA Whole blood Standarg glucose

Sample 2,0 ml 0,2 ml -

Standard 2,0 ml 0,2ml Lalu centrifuge

Blanko -

Filtrat 1,0 ml Standard 1,0 ml TCA 1,0 ml O. toluidin 4 ml 4 ml 4 ml Campur, lalu dipanaskan 80o C pada waterbath, dinginkan. Lalu baca pada spektrofotometer = 625 nm

Lain-lain: Cara pembuatan reagen lihat Hawk (buku lama) atau lihat di internet percobaan apa, cara nya dan cara pembuatan reagennya. Janga lupa cek botolnya baik isi maupun kadarnya , rumus dan lainlain

Dari internet Acetic acid 1 N =6% ,

BM = 60,028 g Sediaan ada yang: Acetic acid glacial = 17,4 N = 99,7% kalau buat 6% caranya V1X N1 = V2 XN2 6X 99,7 ml = 99,7 X 6 ml Jadi ambil 6 ml acetic acid glasial, aqua ad 99,7 ml (pengenceran = 99,7/6X) atau 16,6X Kalau 1N berarti 1ml glasial acetic acid, aquadest ad 17,4 ml Atau 1/17,4 X 99,7 % = 5,07% Acetic acid 36% (w/w) Di botol dari pabrik? Acetic acid 99,8% 2,268 kg? berarti =2263,464 : 60,054 = 37,690 mol Volume botol tersebut 2 L? (cek dulu) 37,690: 2 = 18,85M 1M atau 1 N acetic acid = 99,7% X1/18,85 = 5,305%

1.

[PDF]

Microsoft Word - Practical Buffer Prep

90k - Adobe PDF - View as html acid and acetate. And lastly, what does the sodium have to do with the buffer? Well, ... hydroxide and hydrochloric acid. Your object will be to prepare at ... www.tamucc.edu/~plarkin/4101folder/CHEM 4401 L3 Buffer Prep.pdf

2. PASCO : Chemistry : Quantities and Reactions : Buffers


... (hydrochloric acid) and of a strong base (sodium hydroxide) on ... 0.1 M acetic acid (CH3CO2H) 1.0 M hydrochloric acid (HCl) 0.1 M sodium acetate (CH3CO2Na) ... www.pasco.com/chemistry/quantities-and-reactions/buffers.cfm - Cached

3. "A" Standard Solutions: Suppliers of PH Buffers like Acetate Buffer ...


... (Contains: Sodium Acetate, Sodium Chloride, Hydrochloric Acid and Acetone) ... Contains: Sodium Hydroxide and Potassium Iodide (Contains No Azide's) A-162 ... www.redbirdservice.com/catalog23/solutions A.htm - 68k - Cached

4. EXP 18E: Acid/Base Buffers


... of adding sodium acetate to an acetic acid solution, the most ... buffer solution by adding an excess amount of acetic acid to a sodium hydroxide solution. ... pages.towson.edu/debye/chem111/F2000/111exp18acidbase_e.html - Cached

5. EXP 22C,E: Salt Hydrolysis and Buffer Solutions


Instead of adding sodium acetate to an acetic acid solution (the most direct way ... acid and sodium hydroxide solutions used in making the buffer; compare this ... pages.towson.edu/debye/chem111/experiments/111_exp18CEsaltbuffer.html - Cached

6.

[PPT]

Activity Coefficient..>
475k - Microsoft Powerpoint - View as html ... (sodium hydroxide) 0.10M HCl (hydrochloric acid) ... a = 0.025M acetic acid. s = 0.025M sodium acetate. Buffer pH Titrations. 1. Calibrate pH meter ... iris.nyit.edu/~source/Activity Coefficients and Buffer Capacity Tit...

7. CHEM 215 L Preparation of a Buffer


a. Make a pH = 3.00 buffer using citric acid and sodium hydroxide. b. Make a pH = 5.00 buffer using sodium acetate and hydrochloric acid. ... campbell.edu/faculty/bryan/CHEM215/Lab_Handouts/Buffer_Preparation.htm - Cached

CHEM 215 L Preparation of a Buffer (Revised 11/2004)

Supplemental Reading:
Read Chapter 10 and 11 in Quantitative Analysis by Harris (6<sup>th edition) for more details on buffer preparation. See Appendix G, Harris for various K<sub>a Values. Manual for pH meter : http://www.denverinstrumentusa.com/media/pdf/op-man-basic-ph-revg.pdf (see link on CHEM 215 Webpage)

Introduction: Buffers are solutions which contain reasonable amounts of a weak conjugate acid-base pair. A buffer solution has the ability to resist large changes in pH, because when H<sub>3</sub>O+ (aq) or OH- (aq) is added to the solution is converted to the conjugate form of the weak conjugate acid-base pairs by one of the following equations: H<sub>3</sub>O+ (aq) + A- (aq) D HA (aq) + H<sub>2</sub>O (l) OH- (aq) + HA (aq) D</span><b><span style="font-size: 11pt;">A- (aq) + H<sub>2</sub>O (l) The pH of a buffer can be calculated by the Henderson-Hasselbalch equation: pH = pKa + log where
n<sub>B = moles (or mmoles) of the conjugate base n<sub>A = moles (or mmoles) of the conjugate acid pKa = -log (Ka) for the Ka of the conjugate acid

The buffer may be prepared (direct method) by mixing the appropriate moles of conjugate acid (n<sub>A) with the appropriate number of moles of conjugate base

(n<sub>B). If the conjugate acid and base are not both available, then the buffer may be prepared (indirect method) by partial conversion of the conjugate acid to the conjugate base (or vice versa) using a strong acid or base as appropriate. Note: Dilution of the buffer will cause slight changes in pH because of changes in ionic strength. The buffer capacity depends on the actual moles of conjugate acid and base that are present. Objective: Students will calculate and prepare buffers at a specific pHs. Students will apply theoretical knowledge of the Henderson-Hasselbalch equation and activity coefficients to buffer preparation and to pH calculations.

Experimental: (Prepare any three of the following.) Show your calculations to the instructor before proceding. Calculate the number of grams of solid and mL of 1.0 M NaOH or 1.0 M HCl needed to prepare your buffer. a. Make a pH = 3.00 buffer using citric acid and sodium hydroxide.
b. Make a pH = 5.00 buffer using sodium acetate and hydrochloric acid. c. Make a pH = 6.50 buffer using sodium citrate and hydrochloric acid. d. Make a pH = 9.00 buffer using ammonium chloride and sodium hydroxide.

1. Prepare each buffer to be 0.050 M in the conjugate acid, and 100 mL total volume. (Dilute to volume with deionized water.) If both of your reagents are available as a solid, you should calculate the mass of each reagent you are going to mix. If only one reagent is available as a solid, you will have to add 1.00 M NaOH or HCl to create its conjugate form in solution. Show your calculation of the mass of reagent(s) and, if necessary, the volume of strong acid or base you will mix to create your buffer. Show your calculations to the lab instructor before you proceed.

2. Calibrate your pH meter then measure the pH of your solution with a the pH meter, and record the pH in your notebook. How close did you come to your target pH? Can you think of some reasons why the actual pH of your buffer may differ from the theoretical pH?

3. Challenge- Calculate the ionic strength of your solution. See if you can estimate the true pH of your solution using Equation 10-18 (page 190) of the Harris Text.

4. The final step in buffer preparation is to bring the solution to the desired pH by the addition of strong acid or base. Transfer your buffer to a 250 mL beaker and add enough 1.0 M NaOH or HCl from a buret, with magnetic stirring, to bring the pH to the target. The new volume of your buffer is the original 100 mL plus the volume of acid or base you just added. Calculate the new theoretical pH by calculating the new weak base to conjugate acid ratio and using the Henderson-Hasselbalch equation. To get the new ratio, you will need to calculate the moles of strong acid or base you have just added. Compare the new actual and theoretical pH.

Available Reagents:

1.0 M NaOH (aq), 1.0 M HCl (aq), Citric Acid Monohydrate (H<sub>3</sub>Cit H<sub>2</sub>O) , 210.14 g/mole Sodium Acetate Trihydrate (NaC<sub>2</sub>H<sub>3</sub>O<sub>2 3H<sub>2</sub>O), 136.08 g/mole Sodium Citrate Dihydrate (Na<sub>3</sub>Cit 2H<sub>2</sub>O), 294.10 g/mole Ammonium Chloride (NH<sub>4</sub>Cl), 53.49 g/mole See your textbook for various pKas. (Appendix G)

Formulations of Commonly Used Buffers and Media: These are the formulations of buffers we have successfully used over the years. If they are not the same as other formulations the differences are probably not significant. Citric Saline: To make 500ml of 10X solution 50g KCl 22g Sodium Citrate Dissolve in distilled water and bring to 500ml. Sterilize by autoclaving. Dilute to 1X with sterile distilled water and use to remove adherent cells from tissue culture dishes. To do this aspirate off culture media and replace with 1X solution. Let cells sit in the incubator at 37C, and then monitor cells with microscope, generally they are no longer adherent after as little as 5 minutes. Collect cells by centrifugation, replace citrate saline with regular culture media and replate cells. Cheap and Efficient! Mounting Media for Immunofluorescence Microscopy: To make 50mls of add 40g glycerol to 5mls of 10X PBS and make up to 50mls volume. To stain for DNA add Hoechst 33258 dye to a final concentration of 2.5mM. But if you put the Hoechst dye into your media, be very careful, since this dye is a DNA intercalating agent which may well be carcinogenic. Phosphate Buffered Saline: To make 1L of 10X solution 2g KH<sub>2</sub>PO<sub>4 Potassium dihydrogen phosphate, a.k.a. potassium phosphate monobasic 14.1g Na<sub>2</sub>HPO<sub>4 Anhydrous sodium phosphate, a.k.a. sodium phosphate dibasic. 2g KCl Potassium Chloride 80g NaCl Sodium Chloride pH to 7.4 with 5N NaOH. (be careful, concentrated NaOH, perhaps surprisingly given that every lab has some around, is quite dangerous. It can blind you if it gets in your eyes!) 1X PBS is 1.47mM KH<sub>2</sub>PO<sub>4, 10mM Na<sub>2</sub>HPO<sub>4, 2.7mM KCl, 137mM NaCl pH=7.4. It is a more or less physiological buffer which living mammalian cells can tolerate at least for a short time. It is frequently used to wash cells prior to protein extraction or immunostaining. It is also useful for antibody incubations in immunocytochemical staining of cells in tissue culture and sections. One caveat is that phosphatase based detection systems are inhibited by high concentrations of phosphate, the phosphatase reaction product, so you need to do a wash in some sort of

phosphate free buffer before you use these. For antibody staining and washing you can also add 0.1% Tween 20 (Polyoxyethylene 20-sorbitol monolaurate) or 0.1% Triton X-100 non-ionic detergents to reduce background staining. Tris Buffered EDTA (TBE): To make 1L of 10X solution 107.8g Tris base ~55g Boric acid 7.44g EDTA add less than total amount of Boric acid, dissolve this and the Tris in 800 ml of distilled water and make pH to 8.3 by adding more boric acid. Finally make to 1L final volume. This is the standard buffer for running DNA in agarose gels. Tris Buffered Saline: To make 1L of 10X solution 12.1g Tris base 87.66g NaCl pH with concentrated HCl (be careful!) to 7.5 1X TBS is 10 mM Tris/HCl, 150 mM NaCl, pH=7.5. We use TBS routinely for ELISA, immunoblots etc. Can also add 0.1% Tween 20 or 0.1% Triton X-100 non-ionic detergents to final concentration of 0.1% reduce background. EnCor Biotechnology Inc. 2007-2008. homepage press www.encorbio.com. Phosphate buffered saline From Wikipedia, the free encyclopedia Jump to: navigation, search Phosphate buffered saline (abbreviated PBS) is a buffer solution commonly used in biological research. It is a salty solution containing sodium chloride, sodium phosphate, and (in some formulations) potassium chloride and potassium phosphate. The buffer helps to maintain a constant pH. The osmolarity and ion concentrations of the solution usually match those of the human body (isotonic). Contents

To go to the EnCor

1 Applications 2 Preparation 3 References

4 External links

[edit] Applications PBS has many uses because it is isotonic and non-toxic to cells. It can be used to dilute substances. It is used to rinse containers containing cells. PBS can be used as a diluent in methods to dry biomolecules, as water molecules within it will be structured around the substance (protein, for example) to be 'dried' and immobilized to a solid surface[citation needed]. The thin film of water that binds to the substance prevents denaturation or other conformational changes. Carbonate buffers may be used for the same purpose but with less effectiveness[citation needed]. PBS can be used to take a reference spectrum when measuring the protein adsorption in ellipsometry[citation needed]. Additives can be used to add function. For example, PBS with EDTA is also used to disengage attached and clumped cells. Divalent metals such as zinc, however, cannot be added as this will result in precipitation. For these types of applications, Good's buffers are recommended. [edit] Preparation There are many different ways to prepare PBS. Some formulations do not contain potassium, while others contain calcium or magnesium[1]. One of the most common preparations is described below. The simplest way to prepare a PBS solution is to use PBS buffer tablets. They are formulated to give a ready to use PBS solution upon dissolution in a specified quantity of distilled water. They are available in the standard volumes: 100, 200, 500 and 1000 ml [2]. A 10 liter stock of 10x PBS can be prepared by dissolving 800 g NaCl, 20 g KCl, 144 g Na<sub>2</sub>HPO<sub>4 and 24 g KH<sub>2</sub>PO<sub>4 in 8 L of distilled water, and topping up to 10 L. The pH is ~6.8, but when diluted to 1x PBS it should change to 7.4. When making buffer solutions, it is good practice to always measure the pH directly using a pH meter. If necessary, pH can be adjusted using hydrochloric acid or sodium hydroxide. On dilution, the resultant 1x PBS should have a final concentration of 137 mM NaCl, 2.7 mM KCl, 10 mM Sodium Phosphate dibasic, 2 mM Potassium Phosphate monobasic and a pH of 7.4. Another preparation is described in Molecular Cloning by Sambrook, Fritsch and Maniatis, Apendix B.12[3] as follows:

For 1 litre of 1x Phosphate-buffered saline (1x PBS buffer) use:.. - Dissolve in 800 ml of distilled H<sub>2</sub>O: - 8 g of NaCl - 0.2 g of KCl - 1.44 g of Na<sub>2</sub>HPO<sub>4 - 0.24 g of KH<sub>2</sub>PO<sub>4 - Adjust the pH to 7.4 with HCl or NaOH - Add H<sub>2</sub>O to 1 liter. Dispense the solution into aliquots and sterilize them by autoclaving (20 min, 121C, liquid cycle). Store at room temperature. [edit] References 1. ^ Dulbecco, R. et al. (1954): Plaque formation and isolation of pure lines with poliomyelitis viruses. In: J. Exp. Med. vol. 99 (2), pp. 167-182. PMID 13130792 2. ^ Medicago AB, (2003) Phosphate buffered saline pH 7.4 specification sheet 3. ^ Sambrook, Fritsch, and Maniatis (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, volume 3, appendix B.12

2.2.1 Definition of a Buffer


A buffer is a solution containing substances which have the ability to minimise changes in pH when an acid or base is added to it .
1

A buffer typically consists of a solution which contains a weak acid HA mixed with the salt of that acid & a strong base eg NaA. The principle is that the salt provides a reservoir of A- to replenish [A-] when A- is removed by reaction with H+.

The Major Body Buffer Systems

Site ISF

Buffer System Bicarbonate Phosphate Protein

Comment For metabolic acids Not important because concentration too low Not important because concentration too low Important for metabolic acids Important for carbon dioxide Minor buffer Concentration too low Important buffer Important buffer Responsible for most of 'Titratable Acidity' Important - formation of NH<sub>4+ In prolonged metabolic acidosis

Blood Bicarbonate Haemoglobin Plasma protein Phosphate ICF Proteins Phosphates Urine Phosphate Ammonia Bone Ca carbonate

BUFFERS The quality of fixation is influenced by pH and the type of ions present. The choice of buffer is based on: 1. 2. the buffering capacity in the desired pH range with the ability to maintain constant pH during fixation. the side effects which vary with the tissue type: a. b. c. suitable osmolarity so that cells and organelles neither swell nor shrink during fixation. suitable ionic concentration so that materials are neither extracted nor precipitated during fixation. the toxicity of the buffer.

Criteria of a good buffer: 1. 2. 3. 4. 5. 6. 7. pKa: usually between 6 and 8 desired for biological specimens. Maximum solubility in water and minimum solubility in all other solvents. Reduced ion effects. Dissociation of buffer least influenced by buffer concentration, temperature and ionic composition. Resistance to oxidation (stable). Inexpensive and easy to prepare. No reaction with fixation.

Common Buffers

I.

Phosphate Buffer (Sorenson's buffer) pH 5.8-8

Advantages: 1. 2. 3. 4. Most physiological of common components of extracellular fluids. Non-toxic to cells. pH changes little with temperature. Stable for several weeks at 4 C. buffers. Mimics certain

Disadvantages: 1. Precipitates more likely to occur during fixation. Tends to form precipitates in presence of calcium ions. Precipitates uranyl acetate and tends to react with lead salts. Becomes slowly contaminated with micro-organisms

2.

Preparation of Buffer Stock solutions:


0.2M dibasic sodium phosphate 1 liter Na<sub>2</sub>HPO<sub>4*2H<sub>2</sub>0 35.61 gm or Na<sub>2</sub>HPO<sub>4*7H<sub>2</sub>0 53.65 gm or Na<sub>2</sub>HPO<sub>4*12H<sub>2</sub>0 71.64 gm (MW = (MW = (MW =

178.05)

268.07)

358.14)

+ make

ddH<sub>2</sub>0 1 liter

to

0.2M monobasic sodium phosphate 1 litter

138.01) or

NaH<sub>2</sub>PO<sub>4*H<sub>2</sub>0 27.6 gm

(MW

156.03)

NaH<sub>2</sub>PO<sub>4*2H<sub>2</sub>0 31.21 gm

(MW

+ ddH<sub>2</sub>0 to make liter

Working buffer: 0.1M 100 ml Mix X ml of 0.2M dibasic sodium phosphate with Y ml monobasic sodium phosphate. Dilute to 100 ml with ddH<sub>2</sub>0 or dilute 1:1 with fixative. pH ml (25 Y ml C) X

5.8 4.0 6.0 6.15 6.2 9.25 6.4 13.25 6.6 18.75 6.8 24.5 7.0 30.5 19.5 25.5 31.25 36.75 40.75 43.75 46.0

7.2 36.0 7.4 40.5 7.6 43.5 7.8 45.75 8.0 47.35 2.65 4.25 6.5 9.5 14.0

Osmolarity is adjusted by varying the molarity of phosphates or by the addition of sucrose, glucose or sodium chloride.

At pH 7.2:

0.10M = 226 mOs (milliosmoles) 0.05M = 118 mOs 0.075 = 180 mOs 0.15M = 350 mOs

II.

Cacodylate Buffer (arsenate buffer) pH 5-7.4 Advantages: 1. 2. 3. Easy to prepare. Stable during storage for long periods of time. Does not support growth of microorganisms.

4.

Precipitates usually do not occur. Precipitates do not occur at low concentrations of calcium.

Disadvantages: 1. 2. Toxic. Contains arsenic. Unpleasant smell.

Preparation of Buffer: Stock solutions: 0.2M sodium cacodylate 1 liter


195.92) Na(CH<sub>3)2</sub>As0<sub>2*3H<sub>2</sub>0 42.8 gm + make ddH<sub>2</sub>0 1 liter (MW =

to

0.2M HC1 Conc. HC1 (36-38%) 10 ml ddH<sub>2</sub>0 603 ml

Working buffer: 0.1M

100 ml

Adjust 50 ml of 0.2M sodium cacodylate to desired pH with 0.2M HC1. Dilute to 100 ml with ddH<sub>2</sub>0 or dilute 1:1 with fixative.

pH

0.2M HC1 (ml)

6.4

18.3

6.6 6.8 7.0 7.2 7.4

13.3 9.3 6.3 4.2 2.7

Buffer may also be made with cacodylic acid. Stock solutions: 0.2M cacodylic acid 1 liter 138.0) make (CH<sub>3)2</sub>AsO<sub>2</sub>H 27.6 gm + ddH<sub>2</sub>0 1 liter (MW = to

0.2M NaOH 40) make NaOH 0.8 gm +

100 ml (MW ddH<sub>2</sub>0 100 ml = to

Working buffer: 0.1M Adjust 50 ml of 0.2M cacodylic acid to desired pH with 0.2M NaOH. Dilute to 100 ml with ddH<sub>2 or dilute 1:1 with fixative.

III. Veronal-acetate Buffer Advantages:

(Michaelis buffer)

Useful for block staining with uranyl acetate since precipitates do not form. Disadvantages: 1. 2. 3. 4. Reacts with aldehydes. Poor buffer at physiological pH. Supports growth of micro-organisms. Contains barbiturate.

Preparation of Buffer:

Stock solution: 0.28M

100 ml

Sodium veronal (barbitone sodium)

sub>Na

C<sub>8</sub>H<sub>11</sub>0<sub>3</sub>N<sub>2</ (MW = 206.18) 2.89 gm

Sodium acetate (anhydrous) 82.03) or Sodium acetate (hydrated) 136.09) CH<sub>3</sub>C00Na*3H<sub>2</sub>0 1.90 gm (MW = CH<sub>3</sub>C00Na 1.15 gm (MW =

+ make

ddH<sub>2</sub>H<sub>2</sub>0 100 ml

to

Solution is stable and may be stored for some months at 4 C.

Working buffer:

Veronal acetate stock solution ddH<sub>2</sub>0 15 ml

5 ml

Add 0.1 HC1 gradually to desired pH.

Solution cannot be stored. Supports growth of bacteria and molds even at 4 C. Crystallizes in absence of osmium tetroxide.

IV.

Collidine Buffer

pH 7.25-7.74

Advantages:

1.

Maximum buffering capacity about 7.4.

2.

Stable indefinitely at room temperature.

3.

Useful for fixation of large tissue blocks. Aids penetration of fixative due to extractive effects (see disadvantage 1).

Disadvantages:

1.

Not suitable as buffer during primary fixation with osmium tetroxide due to considerable extraction of tissue components.

2.

Use leads to lysis of cytoplasmic matrix and extensive membrane destruction when used with paraformaldehyde fixatives.

3.

Use gives poorer results with glutaraldehyde than those obtained with phosphate or cacodylate buffer.

Preparation of Buffer:

Stock solution:

0.4M

100 ml

Pure collidine 5.34 gm 2,4,6(CH<sub>3)3(C<sub>2</sub>H<sub>5</sub>N) (MW = 121.18)

s-

+ make

ddH<sub>2</sub>0 100 ml

to

Working buffer:

0.2M

100 ml

Adjust 50 ml of s-collidine stock solution to desired pH with 1N HC1. Dilute to 100 ml with ddH<sub>2</sub>0.

pH

1N HC1 (ml)

7.25 7.33 7.41 7.5 7.59 7.67 7.74

22 20 18 16 14 12 10

V.

Tris buffer

Advantages:

1.

Good buffering capacity at higher pH required for some tissues and some cytochemical procedures.

2.

"More or less" physiologically inert.

Disadvantages:

1.

pH changes with temperature. Must be measured at desired temperature.

2.

pH must be measured with certain type of electrode.

Preparation of Buffer:

A.

Tris Buffer

pH 7.1-8.9

Stock solution

0.2M

1 liter

Tris(hydroxymethyl)aminomethane 24.2 gm H<sub>2</sub>NC(CH<sub>2</sub>0H)3 (MW = 121.13)

+ make

ddH<sub>2</sub>0 1 liter

to

Working buffer: 0.1M

100 ml

Adjust pH of 50 ml of stock solution with 0.1M NaOH. Dilute to 100 ml with ddH<sub>2</sub>0.

B.

Tris-maleate Buffer

pH 5.8-8.2

Stock solution: 0.2M

liter

Tris(hydroxymethyl)aminomethane 24.2 gm

Maleic acid (MW = 116.07) 23.2 gm HO<sub>2</sub>CCH:CHCO<sub>2</sub>H

+ make or 237.2) make Trizima-maleate 47.4 gm +

ddH<sub>2</sub>0 1 liter

to

(MW ddH<sub>2</sub>0 1 liter

= to

Working buffer: 0.2M

100 ml

Adjust 50 ml of stock solution to desired pH with 0.1M NaOH. Dilute to 100 ml with ddH<sub>2</sub>0.

VI.

Special Buffers Used for Cytochemical Reactions.

A.

Acetate Buffer 5.6

(sodium acetate-acetic acid buffer) pH 4-

Sodium acetate

0.2M = 27.2 gm/1 (MW -

CH<sub>3</sub>CO<sub>2</sub>Na*3H<sub>2</sub>0 136.09)

Acetic acid

0.2M (MW = 60)

CH<sub>3</sub>COOH

Add sodium acetate to acetic acid to give desired pH. Dilute with ddH<sub>2</sub>0 to desired molarity.

B.

Borate Buffer

pH 7.4-9.2

Borax (sodium tetraborate)

0.2M = 76.2 gm/ml

Na<sub>2</sub>B<sub>4</sub>0<sub>7*120H<sub>2</sub>0 (MW = 381.37)

Boric acid

0.2M = 12.37 gm/1 (MW = 61.83)

H<sub>3</sub>BO<sub>3

Add boric acid to borax solution until desired pH is reached. Dilute to desired molarity with ddH<sub>2</sub>0.

C.

Citrate Buffer (sodium citrate-citric acid buffer) pH 3-6.2

Sodium citrate

0.2M = 58.8 gm/1

Na<sub>3</sub>C<sub>6</sub>H<sub>5</sub>0<sub>7*H<sub>2</sub>0 (MW = 294.12)

Citric acid

0.2M = 42.02 gm/1

C<sub>6</sub>H<sub>8</sub>0<sub>7*H<sub>2</sub>0 (MW = 210.14)

Mix citric acid and sodium citrate to give desired pH. Dilute with ddH<sub>2</sub>0 to desired molarity.

D.

Dimethylglutarate Buffer

pH 3.2-7.6

Dimethylglutaric acid

0.1M = 16.02 gm/1

C<sub>7</sub>H<sub>12</sub>0<sub>4 (MW = 160.2)

Add 0.2N NaOH to give desired ddH<sub>2</sub>0 to desired molarity.

pH.

Dilute

with

E.

Succinate Buffer

pH 3.8-6

Succinic acid

0.2M = 23/62 g/1 (MW = 118.09)

C<sub>4</sub>H<sub>6</sub>0<sub>2

Add 0.2M NaOH to desired pH. Dilute with ddH<sub>2</sub>0 to desired molarity.

F.

Maleate Buffer (sodium hydrogen maleate buffer) pH 5.2-6.8

Stock solution: 0.2M 1 liter

Maleic acid (MW = 121.14) gm + make ddH<sub>2</sub>0 1 liter

23.2 to

Adjust pH with 0.1M Na0H. Dilute with ddH<sub>2</sub>0 to desired molarity.

G.

Imidazole Buffer pH 6.2-7.8

Imidazole 68.08)

0.2M = 13.62/1 (MW =

C<sub>3</sub>H<sub>4</sub>N<sub>2

Adjust 0.2N HC1 to imidazole solution until desired pH is reached. Dilute to desired molarity with ddH<sub>2</sub>0.

H.

AMPd Buffer

pH 7.8-9.7

2-amino-methyl-1,3-propanediol 0.2M = 21.03 gm/1 C<sub>4</sub>H<sub>11</sub>NO<sub>2 (MW = 105.14)

Add 0.2M HC1 until desired pH is reached. Dilute with ddH<sub>2</sub>0 to desired molarity.

Preparation of pH buffer solutions The different names for phosphate salts. Standardization buffers pH 4 and pH 7. Ph range of some buffer systems. Making up buffer solutions by adding an adjuster solution (acid or base) to a known volume and concentration of a primary salt solution. Potassium hydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, potassium hydrogen phthalate, sodium acetate,sodium tetraborate, tris aminomethane. Related link: Analysis Buffers For EDTA titrations For Fluoride (TISAB) For Determination of Fe. On this page: Click the links below to jump to the relevant info:

The preparation of pH buffer solutions acetate buffers phosphate buffers solid mixture buffers Phosphates

Phosphate salts are known by several names and the correct phosphate must be used to prepare buffer solutions. One phosphate cannot be substituted for another phosphate. Check formula of salt to be certain. Formula Name of salt Other names

KH2PO4

potassium dihydrogen orthophosphate monobasic potassium phosphate potassium dihydrogen monopotassium phosphate phosphate acid potassium phosphate potassium biphosphate dipotassium hydrogen orthophosphate potassium hydrogen dipotassium hydrogen phosphate phosphate dibasic potassium phosphate dipotassium phosphate potassium phosphate tribasic potassium phosphate tripotassium phosphate

K2HPO4

K3PO4

Standardization buffers

For pH=7.00 : Add 29.1 ml of 0.1 molar NaOH to 50 ml 0.1 molar potassium dihydrogen phosphate. Alternatively : Dissolve 1.20g of sodium dihydrogen phosphate and 0.885g of disidium hydrogen phosphate in 1 liter volume distilled water. For pH= 4.00 : Add 0.1 ml of 0.1 molar NaOH to 50 ml of 0.1 molar potassium hydrogen phthalate . Alternatively : Dissolve 8.954g of disodium hydrogen phosphste.12 H2O and 3.4023g of potassium dihydrogen phosphate in 1 liter volume distilled water.

Range of common buffer systems

Buffering system Hydrochloric acid/ Potassium chloride Glycine/ Hydrochloric acid Potassium hydrogen phthalate/ Hydrochloric acid Citric acid/ Sodium citrate Sodium acetate/ Acetic acid Potassium hydrogen phtaalate/ Sodium hydroxide

Useful buffering pH range @ 25C 1.0 - 2.2 2.2 - 3.6 2.2 - 4.0 3.0 - 6.2 3.7 - 5.6 4.1 - 5.9

Disodium hydrogen phthalate / Sodium dihydrogen 5.8 - 8.0 orthophospate Dipotassium hydrogen phthalate / Potassium dihydrogen orthophospate

5.8 - 8.0

Potassium dihydrogen orthophosphate / sodium hydroxide Barbitone sodium / Hydrochloric acid

5.8 - 8.00 6.8 - 9.6

Tris (hydroxylmethyl) aminomethane / Hydrochloric 7.0 - 9.00 acid Sodium tetraborate/ Hydrochloric acid Glycine/ Sodium hydroxide Sodium carbonate/ Sodium hydrogen carbonate Sodium tetraborate/ Sodium hydroxide Sodium bicarbonate / Sodium hydroxide Sodium hydrogen orthophosphate / Sodium hydroxide Potassium chloride/ Sodium hydroxide 8.1 - 9.2 8.6 - 10.6 9.2 - 10.8 9.3 - 10.7 9.60 - 11.0

11.0 - 11.9

12.0 - 13.0

Preparing a Buffer Solution This page gives tabulated info on the preparation of buffers by mixing adjusters with a known volume of the primary salt solution. BUFFERS 1.00 - 9.00 Buffer A : pH 1.0 - 2.2 50 ml 0.2 M KCl + mls of 0.2 M HCl Buffer B : pH 2.2 - 4.00 100 ml 0.1 M potassium hydrogen phthalate + mls of 0.1 M Buffer C : Buffer D : pH 4.10 - 5.90 pH 5.8 - 8.00 100 ml 0.1 M . 100 ml 0.1 potassium M KH2PO4 + hydrogen mls of 0.1 M phthalate + mls NaOH. of 0.1 M NaOH Buffer E : pH 7.0 - 9.00 100 ml 0.1 M tris (hydroxymethyl) aminomethane + mls of 0.1 M HCl.

pH

1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20

HCl. mls of pH mls of 0.2M 0.1M HCl HCl added added 134.0 2.20 99.0 105.6 2.30 91.6 85.0 2.40 84.4 67.2 2.50 77.6 53.2 2.60 70.8 41.4 2.70 64.2 32.4 2.80 57.8 26.0 2.90 51.4 20.4 3.00 44.6 16.2 3.10 37.6 13.0 3.20 31.4 10.2 3.30 25.8 7.8 3.40 20.8 3.50 16.4 3.60 12.6 3.70 9.0 3.80 5.8 3.90 2.8 4.00 0.2

pH

4.10 4.20 4.30 4.40 4.50 4.60 4.70 4.80 4.90 5.00 5.10 5.20 5.30 5.40 5.50 5.60 5.70 5.80 5.90

mls of 0.1M NaOH added 2.6 6.0 9.4 13.2 17.4 22.2 27.2 33.0 38.8 45.2 51.0 57.6 63.2 68.2 73.2 77.6 81.2 84.6 87.4

pH mls of 0.1M NaOH added 5.80 7.2 5.90 9.2 6.00 11.2 6.10 13.6 6.20 16.2 6.30 19.4 6.40 23.2 6.50 27.8 6.60 32.8 6.70 38.6 6.80 44.8 6.90 51.8 7.00 58.2 7.10 64.2 7.20 69.4 7.30 74.0 7.40 78.2 7.50 82.2 7.60 85.6 7.70 88.4 7.80 90.6 7.90 92.2 8.00 93.4

pH

mls of 0.1 M HCl added

7.00 7.10 7.20 7.30 7.40 7.50 7.60 7.70 7.80 7.90 8.00 8.l0 8.20 8.30 8.40 8.50 8.60 8.70 8.80 8.90 9.00

93.2 91.4 89.4 86.8 84.0 80.6 77.0 73.2 69.0 64.0 58.4 52.4 45.8 39.8 34.4 29.4 24.4 20.6 17.0 14.0 11.4

BUFFERS 08 - 13

Buffer F: pH 8.0 - Buffer G : 9.10 pH 9.2 - 10.80

Buffer H : pH 9.60 11.00 100 mL 0.025 M 100 mL 0.025 M 100 mL 0.05 Na2B4O7.10H2O Na2B4O7.10H2O M NaHCO3 (borax) + mls of (borax) + mls of + mls of 0.1 0.1 M HCl. 0.1 M NaOH. M NaOH. pH mls of 0.1M pH HCl added mls of 0.1M NaOH added 1.8 7.2 12.4 17.6 22.2 26.2 30.0 33.4 36.6 39.0 41.0 42.6 44.2 45.4 46.6 47.6 48.5 pH mls of 0.1M NaOH added 10.0 12.4 15.2 18.2 21.4 24.4 27.6 30.4 33.0 35.6 38.2 40.4 42.4 44.0 45.4

Buffer I : pH 10.90 12.00 100 mL 0.05 M Na2HPO4 + mls of 0.1 M NaOH. pH mls of 0.1M NaOH added 6.6 8.2 10.2 12.6 15.2 18.2 22.2 27.0 32.4 38.8 46.0 53.8

8.00 8.10 8.20 8.30 8.40 8.50 8.60 8.70 8.80 8.90 9.00 9.10

41.0 39.4 37.6 35.4 33.2 30.4 27.0 23.2 19.2 14.2 9.2 4.0

9.20 9.30 9.40 9.50 9.60 9.70 9.80 9.90 10.00 10.10 10.20 10.30 10.40 10.50 10.60 10.70 10.80

9.60 9.70 9.80 9.90 10.00 10.10 10.20 10.30 10.40 10.50 10.60 10.70 10.80 10.90 11.00

10.90 11.00 11.10 11.20 11.30 11.40 11.50 11.60 11.70 11.80 11.90 12.00

Buffer J : pH 12.00 13.00 50 mL 0.2 M KCl + volume indicated (in mL) 0.2 M NaOH. pH mls of 0.2M NaOH added 12.00 12.0 12.10 16.0 12.20 20.4 12.30 25.6 12.40 32.4 12.50 40.8 12.60 51.2 12.70 64.4 12.80 82.4 12.90 106.0 13.00 132.0

Acetate buffer solutions pH 3 - 6 Make up the following solutions (1) 0.1M acetic acid

(2) 0.1M sodium acetate (tri-hydrate) (13.6g / l) Mix in the following proportions to get the required ph ph 3 4 5 6 vol. of 0.1M acetic acid 982.3 mls 847.0 mls 357.0 mls 52.2 mls vol. of 0.1M sodium acetate 17.7 mls 153.0 mls 643.0 mls 947.8 mls

Phosphate buffer solutions ph 7 - 11 Make up the following solutions (1) 0.1M disodium hydrogen phosphate (14.2g / l) (2) 0.1M HCl (3) 0.1M NaOH Mix in the following proportions to get the required ph ph vol. of phosphate vol. of 0.1M HCl vol. of 0.1M NaOH 7 756.0 mls 244 mls 8 955.1 mls 44.9 mls 9 955.0 mls 45.0 mls 10 966.4 mls 33.6 11 965.3 mls 34.7

Addition of acid or base to a salt pH 3 - 11 Here, the primary salt is a solid and is weighed out in grams. A measured amount of 0.1M HCl or NaOH is added, then made up to 1 liter to give the relevant buffer solution 4. pH 3 4 5 Salt mixture Dilute each mixture to 1 liter solution with distilled water 10.21g potassium hydrogen phthalate and 223ml of 0.10M HCl 10.21g potassium hydrogen phthalate and 1ml of 0.10M HCl 10.21g potassium hydrogen phthalate and 226ml of 0.10M NaOH

6 7 8 9

6.81g potassium dihydrogen phOsphate and 56ml of 0.10M NaOH 6.81g potassium dihydrogen phosphate and 291ml of 0.10M NaOH 6.81g potassium dihydrogen phosphate and 467ml of 0.10M NaOH 4.77g sodium tetraborate and 46ml of 0.10M HCl

10 4.77g sodium tetraborate and 183ml of 0.10M NaOH 11 2.10g sodium bicarbonate and 227ml of 0.10M NaOH

1. The Physical and Theoretical Laboratory, Oxford University. 2. "Electrolyte solutions" Robinson, R. A., and Stokes, R. H., 2nd ed., rev. London, Butterworths, 1968. 3. "Practical chemistry" J. Lambert and T.A. Muir, 3rd. Ed. Heineman, London. 4. pdf file, www.bc.ca/bcsc/resources/ (Canadian Teachers Federation). delloyd.50megs.com

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MEDIA, REAGENTS AND SOLUTIONS

REAGENTS
The following reagents are used in the Microbiological Analyses Section of this Volume and are referenced in that Section. The listing is alphabetical. Brilliant green solution Brilliant green dye, sterile: 0.1 g Distilled water, sterile: 100 ml On day of use, add 20 ml I2-KI solution and 10 ml brilliant green solution to 1 litre base. Re-suspend precipitate by gentle agitation and aseptically dispense 10 ml portions into 20 x 150 or 16 x 150 mm sterile test tubes. Do not heat medium after addition of I2-KI and dye solutions. Bromcresol Purple Dye Solution (0.2%) Bromcresol purple dye: 0.2 g Sterile distilled water: 100 ml Butterfield's Phosphate-Buffered Dilution Water KH2PO4: 34 g Distilled water: 500 ml Adjust pH to 7.2 with 1 N NaOH. Bring volume to 1 liter with distilled water. Sterilize 15 min at 121. Store in refrigerator. Dilution blanks Take 1.25 ml of above stock solution and bring volume to 1 litre with distilled water. Dispense into bottles to 90 or 99 1 ml. Sterilize 15 min at 121. Cellulase Solution

Dissolve 1 g cellulase in 99 ml sterile distilled water. Filter sterilize through a 0.45 m filter. Cellulase solution may be stored at 2-5 for 2 weeks. Chlorine Solution (200 ppm) Commercial bleach (5.25% sodium hypochlorite): 8 ml Distilled water containing 1 g sodium dodecyl sulphate: 992 ml Dissolve 1 g sodium dodecyl sulfate in 992 ml distilled water. Add 8 ml commercial bleach and mix well. Make immediately before use. Ethanol Solution (70%) Ethanol (95%): 700 ml Distilled water: add to final volume of 950 ml Formalinized Physiological Saline Solution Formaldehyde solution (36-38%): 6 ml NaCl: 8.5 g Distilled water: 1 litre Dissolve 8.5 g NaCl in 1 liter distilled water. Autoclave 15 min at 121. Cool to room temperature. Add 6 ml formaldehyde solution. Do not autoclave after addition of formaldehyde. Hydrochloride Solution (1 N) HCl (concentrated): 89 ml Distilled water to make 1 litre Kovac's Reagent p-Dimethylaminobenzaldehyde: 5 g Amyl alcohol (normal only): 75 ml HCl (concentrated): 25 ml Dissolve p-dimethylaminobenzaldehyde in normal amyl alcohol. Slowly add HC1. Store at 4. To test for indole, add 0.2-0.3 ml reagent to 5 ml of 24 h bacteria culture in tryptone broth. Dark red colour in surface layer is positive test for indole. For enteropathogenic E. coli, also test at 72 h if negative at 24 h. Lysostaphin Solution Dissolve 2.5 mg of lysostaphin in 0.02M phosphate-saline buffer containing 1 % NaCl.

Methyl Red Indicator Methyl red: 0.1 g Ethanol (95%): 300 ml Distilled water to make 500 ml Nonfat dry milk Nonfat dry milk: 100g Distilled water: 1 litre For Salmonella: Suspend 100 g dehydrated nonfat dry milk in 1 liter distilled water. Swirl until dissolved. Autoclave 15 min at 121. Papain Papain: 5 g Distilled water: 1 litre Add papain to sterile, distilled water and swirl to dissolve completely. Dispense 100 ml portion into bottles. Physiological Saline Solution Sterile (0.85%) NaCl: 8.5 g Distilled water: 1 litre Dissolve 8.5 g NaCl in water. Autoclave 15 min at 121. Cool to room temperature. Potassium Hydroxide Solution (40%) KOH: 40 g Distilled water to make 100 ml Sodium Hydroxide Solution (1 N) NaOH: 40 g Distilled water to make 1 litre Voges-Proskauer (VP) Test Reagents Solution 1

alpha-Naphthol: 5 g Alcohol (absolute): 100 ml Solution 2 Potassium hydroxide: 40 g Distilled water to make 100 ml Voges-Proskauer (VP) test. At room temperature, transfer 1 ml of 48 h culture to test tube and add 0.6 ml solution 1 and 0.2 ml solution 2. Shake after adding each solution. To intensify and speed reaction, add a few creatine crystals to mixture. Read results 4 h after adding reagents. Development of eosin pink colour is a positive.

BUFFER SOLUTIONS
Buffer Test Solutions
Buffer TS (pH 2) Combine 11.90 ml of 0.2 M hydrochloric acid and 88.10 ml of 0.2 M potassium chloride, and dilute to 200 ml with water. Buffer TS (pH 5) Add 51.5 ml of 0.2 M disodium hydrogen phosphate to 48.5 ml of 0.1 M citric acid. Buffer TS (pH 5.45) Dissolve 1.8360 g of citric acid and 3.198 g of disodium hydrogen phosphate in carbon dioxide-free water to make 200 ml. Buffer TS (pH 6.5) Combine 50 ml of 0.2 M potassium dihydrogen phosphate and 15.2 ml of 0.2 M sodium hydroxide, and dilute to 200 ml with water. Buffer acetate TS (pH 5.0) Add 4.6 g of anhydrous sodium acetate to 11.6 ml of 2 M acetic acid and dilute to 200 ml with water. Adjust the pH to 5.0 0.1 with glacial acetic acid or 10% sodium hydroxide solution. Barbital buffer solution (pH 7.6) Dissolve 4.3 g of barbital sodium in 200 ml of water, adjust the pH to 7.6 with dilute hydrochloric acid, and filter.

Citric acid buffer solution Dissolve 21 g of citric acid in water to make 1,000 ml (Solution A). Dissolve 28.4 g of disodium hydrogen phosphate in water to make 1,000 ml (Solution B). Combine 11 volumes of Solution A and 389 volumes of Solution B. Formic acid buffer solution (pH 2.5) Add 18 ml of water to 0.8 ml of formic acid, adjust the pH to 2.5 with strong ammonia TS, and dilute to 200 ml with water. Phosphate buffer solution (pH 7.0) Combine 50 ml of 0.2 M potassium dihydrogen phosphate and 29.54 ml of 0.2 M sodium hydroxide, and dilute to 200 ml with water. Phosphate buffer solution (pH 7.3-7.4) (0.02M) Stock solution 1: Sodium phosphate dibasic anhydrous: 28.4 g NaCl: 85 g Distilled water: 1 litre Stock solution 2: Sodium phosphate monobasic monohydrate: 27.6 g NaCl: 85 g Distilled water: 1 litre Make 1:10 dilutions of each stock solution. For example: Stock solution 1 Distilled water Approximate pH 50 ml 450 ml 8.2 Stock solution 2 Distilled water Approximate pH 10 ml 90 ml 5.6

Using a pH meter, titer diluted solution1 to pH 7.3-7.4 by adding about 65 ml of solution 2. Use the resulting 0.02 M phosphate saline buffer solution in the lysostaphin susceptibility test on S. aureus. Note: Do not titer 0.2 Mphosphate buffer to pH 7.3-7.4 and then dilute to 0.02 Mstrength. This results in a drop in pH of approximately 0.25. Addition of 0.85% salt after pH adjustment also results in a drop of approximately 0.2. Phosphate buffer solution (pH 7.5)

Dissolve 53.7 g of disodium hydrogen phosphate in water to make 1,000 ml (Solution A). Dissolve 20.4 g of potassium dihydrogen phosphate in water to make 1,000 ml (Solution B). Combine 21 volumes of Solution A and 4 volumes of Solution B, and adjust the pH to 7.5 with either Solution A or Solution B.

Standard Buffer Solutions


Reagent Solutions Previously dry the crystalline reagents (except for boric acid), at 110 to 120, and use water that has been previously boiled and cooled to prepare the solutions. Store the prepared reagent solutions in chemically resistant glass or polyethylene bottles, and use within 3 months. Discard if moulding is evident.

Boric acid/potassium chloride, 0.2 M Dissolve 12.366 g of boric acid (H3BO3) and 14.911 g of potassium chloride (KC1) in water to make 1,000 ml.

Hydrochloric acid, 0.2 M Dilute 19 ml of hydrochloric acid with water to make 1,000 ml and standardize the solution as follows: dissolve about 0.3 g, accurately weighed, of primary standard anhydrous sodium carbonate (Na2CO3), previously dried at about 270 for 1 h in 100 ml of water. Titrate with the hydrochloric acid using 2 drops of methyl red TS. When the solution becomes faintly pink, boil to expel carbon dioxide, cool, and continue the titration until the faint pink colour is no longer affected by continued boiling. Each 10.60 mg of Na2CO3 is equivalent to 1 ml of 0.2 M hydrochloric acid.

Potassium chloride, 0.2 M Dissolve 14.911 g of potassium chloride (KC1) in water to make 1,000 ml.

Potassium hydrogen phthalate, 0.2 M Dissolve 40.844 g of potassium hydrogen phthalate [KHC6H4(COO)2] in water to make 1,000 ml.

Potassium dihydrogen phosphate, 0.2 M Dissolve 27.218 g of potassium dihydrogen phosphate (KH2PO4) in water to make 1,000 ml.

Sodium hydroxide, 0.2 M

Dissolve about 9 g of sodium hydroxide (NaOH) in about 950 ml of water, and add a freshly prepared saturated solution of barium hydroxide until no more precipitate forms. Shake the mixture thoroughly, and allow it to stand overnight in a stoppered bottle. Decant or filter the solution, and standardize the clear liquid as follows: Dissolve about 1 g, accurately weighed, of primary standard potassium hydrogen phthalate [KHC6H4(COO)2], previously dried at 105 for 3 h in 75 ml of carbon dioxide-free water, and titrate with the sodium hydroxide solution to a permanent pink colour using 2 drops of phenolphthalein TS, as indicator. Each 40.84 mg of KHC6H4(COO)2 is equivalent to 1 ml of 0.2 M sodium hydroxide. Composition of Standard Buffer Solutions To prepare a standard buffer solution having a pH within the range 1.2 to 10.0, combine the appropriate solutions, prepared above, as shown in the following table, and dilute with water to make 200 ml. The standard pH values given in this table are considered to be reproducible to within 0.02 of the pH unit specified at 25. Acid Phthalate Neutralized Phosphate Alkaline Borate Buffer Phthalate Buffer Buffer Buffer To 50.0 ml of 0.2 To 50.0 ml of 0.2 To 50.0 ml of 0.2 To 50.0 ml of 0.2 To 50.0 ml of 0.2 M KHC6H4M KHC6H4 M H3BO3KCl M KCl add the M KH2PO4 add (COO)2 add the (COO)2 add the add the specified specified ml of the specified ml specified ml of specified ml of ml of 0.2 M 0.2MHC1 of 0.2 M NaOH 0.2MHC1 0.2 M NaOH NaOH pH ml pH ml pH ml pH ml pH ml 1.2 85.0 2.2 49.5 4.2 3.0 5.8 3.6 8.0 3.9 1.3 67.2 2.4 42.2 4.4 6.6 6.0 5.6 8.2 6.0 1.4 53.2 2.6 35.4 4.6 11.1 6.2 8.1 8.4 8.6 1.5 41.4 2.8 28.9 4.8 16.5 6.4 11.6 8.6 11.8 1.6 32.4 3.0 22.3 5.0 22.6 6.6 16.4 8.8 15.8 1.7 26.0 3.2 15.7 5.2 28.8 6.8 22.4 9.0 20.8 1.8 20.4 3.4 10.4 5.4 34.1 7.0 29.1 9.2 26.4 1.9 16.2 3.6 6.3 5.6 38.8 7.2 34.7 9.4 32.1 2.0 13.0 3.8 2.9 5.8 42.3 7.4 39.1 9.6 36.9 2.1 10.2 4.0 0.1 7.6 42.4 9.8 40.6 2.2 7.8 7.8 44.5 10.0 43.7 8.0 46.1 Hydrochloric Acid Buffer

STANDARD SOLUTIONS
Ammonium Standard Solution

Dissolve 296.0 mg of ammonium chloride, NH4C1, in sufficient water to make 100 ml. Transfer 10.0 ml of this solution into a 1,000-ml volumetric flask, dilute to volume with water. Each ml of this solution contains 0.01 mg of NH+4. Barium Standard Solution Dissolve 177.9 mg of barium chloride, BaCl2.2H20, in water in a 1,000-ml volumetric flask, dilute to volume with water, and mix. Each ml of this solution contains 0.1 mg of Ba. Barium Chloride Standard Solution Dissolve 4.3 g of barium chloride in sufficient water to make 1,000 ml. Perform gravimetric analysis on the solution, and calculate the quantity of sodium sulfate (Na2SO4) corresponding to 1 ml of the solution. Each ml of this solution corresponds to about 2.5 mg of Na2SO4. Chromium Standard Solution To 0.934 g of potassium chromate, add 1 drop of 10% sodium hydroxide solution and water to 1,000 ml. To a 1.0 ml portion of the solution, add 1 drop of 10% sodium hydroxide solution and water to 1,000 ml. Each ml of this solution contains 0.25 g of Cr. Condensed Formaldehyde Standard Solution Dilute 8.1 g of formalin (containing 37% of HCHO) with water to 1,000 ml. To a 10.0 ml portion of the solution, add water to 1,000 ml. Each ml of this solution contains 0.03 mg of HCHO. Prepare freshly before use. Dithizone Standard Solution Dissolve 10 mg of dithizone in 1,000 ml of chloroform. Store in a stoppered bottle lead free and in a cold place. Formaldehyde Standard Solution Dilute 2.7 g of formalin (containing 37% of HCHO) with water to 1,000 ml. To a 10 ml portion of the solution, add water to 1,000 ml. Each ml of this solution contains 0.01 mg of HCHO. Prepare the solution fresh. Iron Standard Solution Dissolve 8.63 g of ferric ammonium sulfate in 20 ml of dilute nitric acid, and add water to 1,000 ml. To 10 ml of the solution add 20 ml of dilute nitric acid and water to 1,000 ml. Each ml of this solution contains 0.01 mg of Fe. Store in a dark bottle.

Lead Standard Solution Dissolve 159.8 mg of lead nitrate in 10 ml of dilute nitric acid, and add water to 1,000 ml. Prepare and store this solution in lead-free glassware. Dilute 10 ml of the solution with water to 100 ml. Each ml of this solution contains 0.01 mg of Pb. Prepare the solution fresh. Lead Standard Solution for Dithizone test To 10 ml of lead standard solution, add 1% nitric acid to 100 ml. Each ml of this solution contains 1 g of Pb. Prepare the solution fresh. Magnesium Standard Solution Dissolve 50.0 mg magnesium metal, Mg, in 1 ml of hydrochloric acid in a 1,000-ml volumetric flask, dilute to volume with water, and mix. Each ml of this solution contains 0.05 mg Mg. Mercury Standard Solution Dissolve 0.135 g of mercuric chloride in 10 ml of dilute nitric acid and sufficient water to make 1,000 ml. Dilute 10 ml of the solution with 10 ml of dilute nitric acid and water to make 1,000 ml. Dilute the second solution in same manner. Each ml of this final solution contains 0.1 g of Hg in 1 ml. Prepare the solution fresh. Methanol Standard Solution To 5 ml of 0.1% methanol, add 2,5 ml of ethyl alcohol not containing methanol, and add water to 50 ml. Each ml of this solution contains 0.1 mg of CH3OH. Nitrate Standard Solution Dissolve 1.63 g of potassium nitrate in water to make 1,000 ml. To a 10 ml portion of the solution, add water to 100 ml. Each ml of this solution contains 0.1 mg of NO3. Phosphate Standard Solution Dissolve 143.3 mg of monobasic potassium phosphate, KH2PO4, in water in a 100 ml volumetric flask, dilute to volume with water, and mix. Transfer 10.0 ml of this solution into a 1,000-ml volumetric flask, dilute to volume with water, and mix. Each ml of this solution contains 10 g phosphate. Potassium Phosphate, Monobasic, Standard Solution Dissolve 4.394 g of potassium phosphate monobasic in sufficient water to make 1,000 ml. Each ml of this solution contains 1 mg of phosphate.

Selenium Standard Solution Add 10 ml of dilute sulfuric acid (1 in 2) to 1 g of selenium. Heat to dissolve, and evaporate to dryness on a water bath. Dissolve the residue in sufficient water to make 1,000 ml. To a 10 ml portion of the solution, add water to 1,000 ml. Each ml of this solution contains 0.01 mg of Se. Thiamine Hydrochloride Standard Solution Dissolve 0.1 g of vitamin B1 hydrochloride reference standard previously dried at 105 for 2 h, in water to make 1,000 ml. To a 10 ml portion of the solution, add water to 1,000 ml. Each ml of this solution contains 1 g of vitamin B1 hydrochloride reference standard. Zinc Standard Solution Dissolve 4.4 g of zinc sulfate in water to make 1,000 ml. To a 10 ml portion of the solution, add water to 1,000 ml. Each ml of this solution contains 0.01 mg of Zn.

TEST SOLUTIONS
Potassium Hydroxide TS A 6.5% w/v solution of potassium hydroxide (KOH) in water (approximately N). Potassium Hydroxide TS, Ethanolic Place a few g (5 to 10) of potassium hydroxide in a 2-litre flask, add 1 to 1.5 L of 95% ethanol and boil on a water bath under reflux condenser from 30 to 60 min. Distil and collect the ethanol. Dissolve 40 g of potassium hydroxide, low in carbonate, in 1,000 ml of the distilled ethanol keeping the temperature below 15.5 while the alkali is being dissolved. This solution should remain clear. Potassium Iodate TS A 0.71% w/v solution of potassium iodate in water. Preserve in the dark. Potassium Iodide TS A 16.5% w/v solution of potassium iodide (KI) in water (approximately N). Store in a light-resistant container.

Potassium Permanganate TS A 1.0% w/v solution of potassium permanganate (KMnO4) in water

paper The juice will be used to run two sub sample determinations of starch on each clone. Starch will be measured using the rapid SPRI iodometric method with minor modifications. Juice (3 ml) will be transferred to two test tubes and placed in a boiling water bath for 10 minutes to completely solubilize the starch. After boiling, the juice will be cooled on ice. After cooling, 2N acetic acid (1.2 ml), 10 % KI (0.25 ml) and KIO3 (2.5 ml) will be added in that order 1. Individual Chemicals and Products List (formerly the Master ... Jenis Berkas: PDF/Adobe Acrobat BAN 360MG (ALPHA AMYLASE). 9157. BAND-ADE SAWING FLUID ...... HYDROCHLORIC ACID,YTTRIUM SALT,KI,KIO3, +. 11338. HYDROCINNAMIC ACID ... www.drs.illinois.edu/css/guidesplans/wasteguide/.../appendixA.pdf - Mirip 2. Full text of "Practical physiological chemistry: A Book Designed ... - [ Terjemahkan laman ini ] Experiments on Enzymes' 1. amylases 1. Demonstration of Salivary Amylase. 4 To 25 cc of a 1 per cent starch paste in a small beaker, add 5 drops of saliva ... www.archive.org/stream/.../practicalphysio00hawkgoog_djvu.txt - Mirip A buffer solution resists changes in pH when acids or bases are added or when dilution occurs. ...

The accuracy of Sigma-Aldrich analytical buffer solutions is ensured by careful choice of the buffer substances, the use of deionized water with extremely low conductivity and subsequent calibration of the pH value with standard buffer solutions to DIN 19266.

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Product No. 31044-1L 31045-1L 31046-1L 31103-1L 33544-1L 33545-1L 33547-1L 33552-1L 33582-1L 33643-1L 33643100ML 33643500ML 33643-5L-VP 33643-10LVP 33646-1L

Description Buffer solution pH 1.0 (20C) hydrochloric acid / potassium chloride Buffer solution pH 2.0 (20C) citric acid / hydrochloric acid / sodium chloride Buffer solution pH 3.0 (20C) citric acid / sodium hydroxide / sodium chloride Acetate buffer solution pH 4.65 sodium acetate / acetic acid Buffer solution pH 5.0 (20C) citric acid / sodium hydroxide Buffer solution pH 6.0 (20C) citric acid / sodium hydroxide Buffer solution pH 8.0 (20C) borax / hydrochloric acid Buffer solution pH 13.0 (20C) glycine / sodium hydroxide / sodium chloride Ammonia buffer solution for complexometry, ammonium chloride / ammonia, pH 10 Buffer solution pH 4.0 (20C) citric acid / sodium hydroxide / sodium chloride Buffer solution pH 4.0 (20C) citric acid / sodium hydroxide / sodium chloride Buffer solution pH 4.0 (20C) citric acid / sodium hydroxide / sodium chloride Buffer solution pH 4.0 (20C) citric acid / sodium hydroxide / sodium chloride Buffer solution pH 4.0 (20C) citric acid / sodium hydroxide / sodium chloride

Buffer solution pH 7.0 (20C) potassium dihydrogen phosphate / disodium hydrogen phosphate 33646Buffer solution pH 7.0 (20C) potassium dihydrogen phosphate / disodium hydrogen 100ML phosphate 33646Buffer solution pH 7.0 (20C) potassium dihydrogen phosphate / disodium hydrogen 500ML phosphate Buffer solution pH 7.0 (20C) potassium dihydrogen phosphate / disodium hydrogen 33646-5L-VP phosphate 33646-10L- Buffer solution pH 7.0 (20C) potassium dihydrogen phosphate / disodium hydrogen VP phosphate 33648-1L Buffer solution pH 9.0 (20C) borax / hydrochloric acid 33648Buffer solution pH 9.0 (20C) borax / hydrochloric acid 100ML 33648Buffer solution pH 9.0 (20C) borax / hydrochloric acid 500ML 33648-5L-VP Buffer solution pH 9.0 (20C) borax / hydrochloric acid 33648-10LBuffer solution pH 9.0 (20C) borax / hydrochloric acid VP 33649-1L-R Buffer pH 10.00 borax / sodium hydroxide solution 33649Buffer pH 10.00 borax / sodium hydroxide solution 100ML-R 33649Buffer pH 10.00 borax / sodium hydroxide solution 500ML-R 33650-1L-R Buffer solution pH 11.0 (20C) boric acid / sodium hydroxide solution / potassium

33650100ML-R 33650500ML-R 33651-1L 33651100ML 82563-1L 82563-50ML 82564-1L 82564-50ML 82565-1L 82565-50ML 82566-1L 82566-50ML 82567-1L 82567-50ML 82568-1L 82568-50ML 82571-1L 82571-50ML 82573-1L 82573-50ML 82574-1L 82574-50ML 82575-1L 82575-50ML 82576-1L 82638-1L

chloride Buffer solution pH 11.0 (20C) boric acid / sodium hydroxide solution / potassium chloride Buffer solution pH 11.0 (20C) boric acid / sodium hydroxide solution / potassium chloride Buffer solution pH 12.0 (20C) di-sodium hydrogen phosphate / sodium hydroxide Buffer solution pH 12.0 (20C) di-sodium hydrogen phosphate / sodium hydroxide solution Buffer solution pH 1.0 (20C) hydrochloric acid ~0.13 M, potassium chloride ~0.050 M Buffer solution pH 1.0 (20C) hydrochloric acid ~0.13 M, potassium chloride ~0.050 M Buffer solution pH 2.0 (20C) hydrochloric acid ~0.0082 M, citric acid ~0.03 M, sodium chloride ~0.061 M Buffer solution pH 2.0 (20C) hydrochloric acid ~0.0082 M, citric acid ~0.03 M, sodium chloride ~0.061 M Buffer solution pH 3.0 (20C) citric acid ~0.040 M, sodium hydroxide ~0.021 M, sodium chloride ~0.060 M Buffer solution pH 3.0 (20C) citric acid ~0.040 M, sodium hydroxide ~0.021 M, sodium chloride ~0.060 M Buffer solution pH 4.0 (20C) citric acid ~0.056 M, sodium hydroxide ~0.068 M, sodium chloride ~0.044 M, sodium azide ~0.05 % Buffer solution pH 4.0 (20C) citric acid ~0.056 M, sodium hydroxide ~0.068 M, sodium chloride ~0.044 M, sodium azide ~0.05 % Buffer solution pH 5.0 (20C) citric acid ~0.096 M, sodium hydroxide ~0.20 M Buffer solution pH 5.0 (20C) citric acid ~0.096 M, sodium hydroxide ~0.20 M Buffer solution pH 6.0 (20C) citric acid ~0.060 M, sodium hydroxide ~0.16 M Buffer solution pH 6.0 (20C) citric acid ~0.060 M, sodium hydroxide ~0.16 M Buffer solution pH 7.0 (20C) sodium hydroxide solution ~0.029 M, potassium dihydrogen phosphate ~0.050 M Buffer solution pH 7.0 (20C) sodium hydroxide solution ~0.029 M, potassium dihydrogen phosphate ~0.050 M Buffer solution pH 8.0 (20C) hydrochloric acid ~0.021 M, borax ~0.013 M Buffer solution pH 8.0 (20C) hydrochloric acid ~0.021 M, borax ~0.013 M Buffer solution pH 9.0 (20C) borax ~0.013 M, hydrochloric acid ~0.0046 M Buffer solution pH 9.0 (20C) borax ~0.013 M, hydrochloric acid ~0.0046 M Buffer solution pH 10.0 (20C) sodium hydroxide ~0.018 M, borax ~0.013 M Buffer solution pH 10.0 (20C) sodium hydroxide ~0.018 M, borax ~0.013 M Buffer solution pH 11.0 (20C) glycine ~0.051 M, sodium hydroxide ~0.049 M, sodium chloride ~0.051 M Buffer solution according to Soerensen pH 6.5 Back to Top

Buffer Solutions, pH 1 - 4

Product # Description 36050 Acetate buffer solution pH 4.65 sodium acetate / acetic acid, solution ready for use 31103 Acetate buffer solution pH 4.65 sodium acetate / acetic acid, solution ready for use 33581 Acetate buffer solution pH 4.6 for complexometry, sodium acetate / acetic acid, solution ready for use 82563 Buffer solution pH 1.0 (20 C) potassium chloride ~0.050 M, hydrochloric acid ~0.13 M 31044 Buffer solution pH 1.0 (20 C) hydrochloric acid / potassium chloride, solution ready for use 82564 Buffer solution pH 2.0 (20 C) hydrochloric acid ~0.0082 M, citric acid ~0.03 M, sodium chloride ~0.061 M 456098 Buffer solution pH 2.0 (20C) 33541 Buffer solution pH 2.0 (20 C) citric acid / hydrochloric acid / sodium chloride, solution ready for use, with fungicide 31045 Buffer solution pH 2.0 (20 C) citric acid / hydrochloric acid / sodium chloride, solution ready for use, with fungicide

82565 Buffer solution pH 3.0 (20 C) sodium hydroxide ~0.021 M, citric acid ~0.040 M, sodium chloride ~0.060 M 33542 Buffer solution pH 3.0 (20 C) citric acid / sodium hydroxide / sodium chloride solution, solution ready for use, with fungicide 31046 Buffer solution pH 3.0 (20 C) citric acid / sodium hydroxide / sodium chloride solution, solution ready for use, with fungicide 82566 Buffer solution pH 4.0 (20C) sodium azide ~0.05 %, sodium hydroxide ~0.068 M, citric acid ~0.056 M, sodium chloride ~0.044 M 33643 Buffer solution pH 4.0 (20 C) with fungicide, citric acid / sodium hydroxide / sodium chloride solution, solution ready for use CATATAN; BUFFER SITRAT DAPAT DIBUAT DENGAN CARA 1. DARI ASAM SITRAT DAN SODIUM SITRAT 2. DARI SODIUM SITRAT DAN HCl CONTOH : BUFFER 0.2 M Na- SITRAT / HCl pH 4.5 (0.2 M sodium citrate/HCl buffer pH 4.5)

Dikutip D.L.Bartel Internet Feb05 PBS UNTUK IMUNOHISTOKIMIA 0.5M PB (PHOSPHATE BUFFER) STOCK 106.5 g dibasic sodium phosphate (anhydrous) 34.5 g monobasic sodium phosphate ( anhydrous) Add the above to 1800 mL distilled water. Bring the final volume to 2 L. When the salts have gone into solution, adjust the pH with sodium hydroxide to between 7.2 - 7.4 This stock is kept at room temperature and is good for several weeks.

5M NaCl 292.2 g NaCl


Add the above to 800 mL distilled water. Bring final volume to 1 L. Let stir thouroughly until salt has completely dissolved. This stock is kept at room temperature and is good for several weeks. 0,1 M PBS (working soluion for immuno) 200 mL 0.5M PB stock 30 mL 5M NaCl stock 770 mL distilled water final PB concentration 0.1 M final NaCl concentration 150 mM

1 L PBS for immnuno washes