Rabu, 11 September 2013

KADAR LARUTAN ATAU KONSENTRASI LARUTAN DI LABORATORIUM

KADAR LARUTAN ATAU KONSENTRASI LARUTAN DI LABORATORIUM

Dalam aktivitas di laboratorium akan kita jumpai larutan dengan berbagai simbol konsentrasi maupun kadarnya. ada beberapa satuan kadar atau konsentrasi larutan yang familiar di gunakan di dalam laboratorium Kimia Farmasi, antara lain :
  1. Molalitas, diberi simbol m, adalah jumlah gram molekul (mol) zat terlarut dalam 1 kg pelarut, molalitas bisa dirumuskan m = mol/1000 gr pelarut.
  2.  Molaritas, diberi simbol M, adalah jumlah gram molekul zat (mol) terlarut yang di larutkan dalam pelarut hingga volume 1 L, molaritas bisa di rumuskan M = mol/Liter.
  3. Normalitas, di beri simbol N, adalah jumlah bobot ekuivalen zat yang di larutkan dalam pelarut hingga volume 1 L, normalitas bisa di rumuskan N = mol x valensi / Liter .
  4. Persen bobot per bobot, di beri simbol % b/b, menyatakan jumlah gram zat dalam 100 gram larutan atau campuran.
  5. Persen bobot per volume, di beri simbol % b/v, menyatakan jumlah gram zat dalam 100 ml larutan.
  6. Persen volume volume, di beri simbol % v/v, menyatakan jumlah ml zat dalam 100 ml larutan.
  7. ppm, menyatakan bagian tiap satu juta, atau bisa di sederhanakan jumlah mg zat terlarut dalam 1 Liter larutan.
  8. ppb, menyatakan bagian tiap satu milyar, atau bisa di sederhanakan jumlah ug zat terlarut dalam 1 Liter larutan.
Di dalam suatu metode analisis sering juga di jumpai satuan turunan seperti : mM yang berarti mili molar, atau uM yang berarti mikro molar.
  • Soal Latihan
  1. Berapa ml HCl pekat yang anda perlukan untuk membuat 1 Liter HCl 0,1 M jika larutan HCl pekat yang ada di laboratorium memiliki kadar 37 % b/b dan masa jenisnya 1,19 kg/Liter ?
  2. Berapa ml H2SO4 pekat yang anda harus pipet untuk membuat 500 ml H2SO4 2 N, jika diketahui larutan H2SO4 pekat stok memiliki kadar 95 % b/b dan masa jenisnya 1,84 kg/liter ?
  3. Berapa gram NaOH murni yang akan anda timbang jika anda akan membuat 250 ml larutan NaOH 0,1 M ?

Selasa, 10 September 2013

FRAKSINASI DENGAN KROMATOGRAFI VAKUM CAIR



FRAKSINASI DENGAN KROMATOGRAFI VAKUM CAIR
Dr. Muhtadi, M.Si

Bahan workshop isolasi chemical marker dari bahan alam di UMS 2009

·         Pada prinsipnya adalah kromatografi kolom yang dipercepat proses elusinya dengan bantuan alat vakum (penghisapan)=> waktu lebih cepat, lazimnya hanya 2 – 3 jam saja.

·         Tujuan KVC, untuk fraksinasi senyawa-senyawa dalam suatu ekstrak (menyederhanakan campuran senyawa)

Tahapan pelaksanaan KVC :
1.    Pencarian eluen yang akan digunakan untuk KVC
Prinsipnya :
·         dicari eluen yang memisahkan noda kromatogram dengan Rf mulai  0,1 hingga Rf = 0,5.
·         untuk menghemat pelarut,  biaya lebih murah & menghindari efek toksik yang terjadi, lazimnya digunakan campuran n-heksana:etilasetat.

2.    Penyiapan kolom KVC
Prinsipnya :
·         Berat sampel yang akan dipisahkan akan menentukan pemilihan diameter kolom KVC yang akan digunakan, dengan arahan ketentuan :
Berat sampel (g)
Diameter kolom KKT (cm)
Silika kolom (g)
Perbandingan sampel : silica kvc
15 – 25
14
150 – 250
1 : 10 – 1 : 15
3 – 5
10
60 – 125
1 : 20 – 1 : 25
1 – 2
7
30 – 80
1 : 30 – 1 : 40

·         Tinggi silica dalam kolom KVC lazimnya hanya 5  cm, hingga tinggi maksimal = diameternya (Silica G 60, Katalog Merck : 7730/7731)
·         Packing kolom dilakukan dengan cara : silica dalam kolom KVC dengan dibantu vakum, ditekan-tekan hingga rata, selanjutnya  dihomogenkan dengan eluen (fase gerak) n-heksana.
Catatan : Kolom telah homogen, jika silica tidak ada yang retak & waktu dialiri eluen merata


3.    Penyiapan sampel dengan impregnasi
·         Sampel dilarutkan dalam pelarut yang paling sesuai (larut dengan sempurna). Jika ada sebagian tidak larut, hendaknya sampel yang tidak larut tersebut dipisahkan terlebih dahulu dari larutan dengan penyaringan biasa. Larutan sampel kemudian dicampurkan dengan silika gel impreg (mesh 30 – 70, Katalog : 7733) dengan perbandingan 1:2 antara sampel dengan silica, dan diuapkan pada tekanan rendah menggunakan rotary epaporator sampai kering (bebas pelarut).
·         Sampel yang sudah di-impregnasi dimasukkan ke dalam kolom KVC, dan pada bagian atas sampel diberi kertas saring untuk menghindari penyebaran ekstrak kedalam eluen.

4.    Fraksinasi
Fraksinasi dilakukan dengan menggunakan eluen yang telah terpilih, jumlah fraksi lazimnya hanya 12 – 20 fraksi untuk fraksinasi tahap pertama. Volume eluen untuk setiap kali elusi tergantung dari diameter kolom KVC, dengan arahan sbb. :
Berat sampel (g)
Diameter kolom KKT (cm)
Volume eluen per-elusi (mL)
15 – 25
14
100 – 175
3 – 5
10
75 – 125
1 – 2
7
50 – 75

5.    Pemekatan fraksi dengan cara evaporasi
Pemekatan fraksi dilakukan untuk tujuan agar saat masing-masing fraksi ditotolkan dalam plat KLT, lebih pekat sehingga mempercepat dalam penotolan & noda kromatogram lebih baik.

6.    Analisis KLT hasil fraksinasi
Masing-masing fraksi hasil KKT ditotolkan pada plat KLT, dibandingkan dengan sampel/ekstrak dan standar (chemical marker standar, jika punya). Eluen yang dapat digunakan untuk analisis KLT adalah eluen yang memberikan nilai Rf 0,2 – 0,8 untuk noda-noda kromatogram yang diamati. Fraksi yang memberikan noda kromatogram tunggal dan memiliki Rf sama dengan standar, menunjukkan senyawa murni hasil isolasi yang sama dengan standarnya, selanjutnya digabungkan.



7.    Penggabungan fraksi
Berdasarkan analisis KLT terhadap fraksi-fraksi, maka fraksi yang memiliki noda-noda kromatogram yang mirip dengan nilai Rf yang sama digabungkan, selanjutnya dievaporasi untuk ditentukan beratnya. Fraksi gabungan yang memiliki noda kromatogram lebih sederhana dengan berat yang signifikan dapat dilanjutkan untuk pemurnian. Atau fraksi gabungan yang memiliki noda kromatogram dari chemical marker yang akan diisolasi, dimurnikan lebih lanjut dengan cara KKT atau kromatografi radial atau KLT preparative.

ATURAN DASAR KESELAMATAN KERJA DI LABORATORIUM



ATURAN DASAR KESELAMATAN KERJA
LABORATORIUM KIMIA FARMASI

  1. Bersihkan dan netralkan segera tumpahan ataupun ceceran bahan kimia .
  2. Tidak makan,minum ataupun membuat makanan dan minuman di Laboratorium Kimia Farmasi.
  3. Tidak merokok di Laboratorium Kimia Farmasi.
  4. Tidak berlari-lari ataupun tergesa-gesa bekerja  di Laboratorium Kimia Farmasi.
  5. Tidak menaruh/menyimpan tas ataupun barang barang lainnya di lantai dan di area jalan di Laboratorium Kimia farmasi.
  6. Selalu memakai/mempersiapkan pakaian pelindung  yang di perlukan
    • Safety goggles ( Kaca mata pelindung )
    • Jas Praktikum
    • Celana panjang
    • Sepatu pelindung tertutup
    • Sarung tangan
    • Masker pelindung pernafasan
  7. Gunakan selalu propipet ataupun karet penghisap untuk menghindari                                                    kontak langsung zat kimia ke Kulit.
  8. Jangan pernah menyimpan bahan kimia ke dalam botol ataupun bekas tempat menyimpan makanan ataupun minuman.
  9. Selalu melabeli botol ataupun tempat menyimpan bahan kimia dengan Nama zat dan label bahan/zat, misal :
·         C untuk zat yang bersifat korosif( mengakibatkan luka bakar )
·         Xi untuk zat yang bersifat mengiritasi
·         Xn untuk zat yang berbahaya
·         O untuk zat bersifat mengoksidasi
·         F untuk zat yang mudah terbakar
·         F+ untuk zat sangat mudah terbakar
·         T untuk zat yang beracun
·         T+ untuk zat yang sangat beracun
·         N untuk bahan yang berbahaya bagi lingkungan
·         E untuk zat yang mudah meledak
  1. Tidak melakukan percobaan yang tidak jelas prosedurnya.
  2. Selalu gunakan penunjuk aliran air  pada sistem pendingin dengan air.
  3. Dalam kasus kebakaran, air pendingin pada sistem pendingin dan sumber arus listrik harus di matikan.
  4. Selalu mengisi buret dibawah tinggi mata praktikan.
  5. Selalu perhatikan kategori bahaya bahan kimia yang dipakai, lihat di :
·         Poster tanda bahaya bahan
·         MSDS, ataupun software chemDAT, safeDAT atau akses ke Intra-Internet ke www.merck.de/www.mallbaker.com
·         Label botol
·         Buku fundamental keselamatan kerja di laboratorium


Yogyakarta, 13 Mei 2009
Mengetahui
Ka.Lab. Kimia Farmasi


(M.Hatta Prabowo M.Si., Apt)

Rabu, 26 Desember 2012

Total Protein Determination By the Lowry Method


Authors: Paul Held Ph. D., Senior Scientist, Applications Dept., BioTek Instruments, Inc.; Janet Hurley Dept. of Molecular Physiology and Biophysics University of Vermont School of Medicine

It's Fast, It's Easy, and It Turns Blue
Quantitation of total protein content of samples is a measurement common to many applications in basic science and clinical research. Here we describe the use of BioTek Instruments ELx808 microplate reader to perform the Lowry method for total protein determination.

Introduction

Quantitation of total protein content is a measurement common to many applications in basic science research and routine clinical laboratory practice. Most biochemical studies that involve the measurement of a biological activity require the normalization of that activity to the protein content. The specific activity of a particular enzymatic activity is of particular importance when proteins are being purified or different samples are being compared. The most utilized methods to assay total protein rely on the reduction of copper in the presence of a chromogenic reagent (1, 2). Regardless of the method of protein determination, laboratories requiring high throughput have often adapted the described protocol to a 96-well format.

Materials and Methods

The assay performed in microplates is essentially a micro Lowry assay (1) that has been adapted to microplates. The reagents can be purchased in a kit (Catalogue No. 690-A: Sigma Chemical, St. Louis MO) or obtained as individual components from the same vendor.
A standard curve was prepared as follows. Bovine serum albumin (BSA) powder was dissolved in distilled water and diluted to a concentration of 1 µg/ml. A series of dilutions (0, 1, 2.5, 5, 10, and 20 µg/well) were made in replicates of 4 with a final volume of 100 µl. Samples were diluted such that they would fall within the BSA standard range (0-25 µg / 100 µl) and 100 µl placed in each well. After standards and samples were diluted and transferred to the microplate, 200 ul of biuret reagent was added to each well and mixed thoroughly with repeated pipeting. Biuret reagent was prepared by mixing 0.5 ml of 1% cupric sulfate with 0.5 ml of 2% sodium potassium tartrate, followed by the addition of 50 ml of 2% sodium carbonate in 0.1 N NaOH. The mixture was then allowed to incubate at room temperature for 10-15 minutes prior to the addition of 20 µl per well of 1.0 N Folin & Ciocalteu's reagent. Samples were mixed immediately with repeated pipeting with each addition. Color was allowed to develop for 30 minutes at room temperature and the absorbance measured at 650 nm and blanked on the water only control. Although in these experiments the plates were read immediately, the reaction was found to be stable for up to an hour.
All absorbance determinations were made using an ELx808 Microplate Reader (BioTek Instruments, Winooski, VT) with the reader controlled by an external PC running KC3 data reduction software (BioTek Instruments, Winooski, VT). Regression analysis and statistics of the curve were performed using KC3.

Results

The absorbance of the Lowry reaction was determined for BSA protein concentrations ranging from 0.0 to 20 µg per well. Over this range the absorbance increased in a hyperbolic fashion. Using KC3 data reduction software (BioTek Instruments), a polynomial non linear equation describing the standard curve can be generated .


Figure 1. Linearity of the assay. Concentration curve from 0 to 20 µg/well of BSA with polynomial regression analysis. Image depicts the screen output from KC3 of a typical standard curve of a Lowry protein assay. Note that the equation describing the regression curve is provided along with statistics concerning the curve.

Although the curve begins to plateau at a protein concentrations of 10 µg/well, determinations can be made with a high level of confidence (r2 = 0.99). Determinations in the lower portion of the curve offer the greatest degree of accuracy with a polynomial fit due to the greater change in signal verses change in protein concentration. As demonstrated in Figure 2, if only low concentrations of protein are assayed (i.e. below 10 µg/well) then a calibration curve determined using linear regression analysis rather than a polynomial analysis can be used with confidence (r2 = 0.99). Routine dilution of each sample would be expected to provide determinations at an appropriate concentration.
The flattening of the absorbance curve observed above the 10 µg level and subsequent loss of the linear increase in absorbance for higher protein concentrations is most likely the result of reagents no longer being in total excess in relation to the oxidizable amino acids necessary for the colorimetric reaction to take place. With high protein levels, reacted chromogenic material was found to precipitate out of solution.


 Calibration curve. Concentration curve from 0 to 10 mg/well of BSA with linear regression analysis. Using the data depicted in Figure 1 a linear regression analysis was performed using the 0-10 µg/well standards. Image depicts the screen output from KC3 of a typical standard curve of a Lowry protein assay. Note that the equation describing the regression curve is provided along with statistics concerning the curve.

Discussion

The ability to easily and reliably quantitate total protein content in samples is paramount to many biological assays. Although the Lowry protein assay was first published in 1951, several improvements, not the least of which is the reduction in assay volume, have increased sensitivity of the assay. Recently fluorescent protein assays have been developed with improved sensitivity (3), but the cost per assay can make them unacceptable for large numbers of samples.
Although the Lowry total protein assay has withstood the test of time, there are several features of the assay that have to be kept in mind. Because these methods rely on the presence of readily oxidizable amino acids such as tyrosine, cysteine, and tryptophan there is a variation in response from proteins with differing amino acid content. Therefore it is advisable that the protein used for generating the standard curve be consistent from experiment to experiment. Likewise, an overabundance of the amino acids in relation to the assay reagents, as would occur with high protein level, will result in a loss of linearity of the assay. In extreme cases this will lead to a precipitation of the chromogens and loss of color prematurely. Likewise, the assay color is only stable for approximately one hour, after which a similar phenomenon occurs in samples with normal concentrations.
The use of KC3 software to control the reader allows the user a great deal of flexibility in regards to data reduction capabilities. The software allows the user to define any configuration of plate map necessary. With several different curve fit algorithms to choose from, regression analysis of the standards and the subsequent concentration determinations of samples can be accomplished with a high degree of confidence. Likewise, the software is capable of performing statistical analysis on sample groups, as well as any mathematical calculation required by the user.
Like most assays that are read in microplates, the ability to read all of the samples simultaneously greatly reduces the manual labor required to obtain the data. The microplate format also lends itself to 'off the shelf' automation for laboratories with high volume requirements. The smaller reaction volumes in microplates will lead to lower per assay costs by reducing the amount of expensive reagents necessary to perform the assay.

References

1. Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall (1951) Protein Measurement with the Folin Phenol Reagent. J. Biol. Chem. 193:265-275
2. Smith, P.K., et al. (1985) Measurement of Protein Using Bicinchoninic Acid. Anal. Biochem. 150:76-85.
3. NanoOrange Protein Quantitation Kit Instructions Molecular Probes, Inc. Eugene Oregon