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Thin Layer Chromatography

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BAB I
PENDAHULUAN

1.1 LATAR BELAKANG
Perkembangan ilmu pengetahuan pada zaman sekarang telah berkembang dengan cukup pesat, terutama di bidang teknologi itu sendiri. Termasuk dalam bidang kedokteran itu sendiri. Pada makalah ini akan dibahas mengenai salah satu teknik ataupun metode yang digunakan dalam bidang kedokteran yaitu kromatografi terutama kromatografi lapis tipis.
Kromatografi adalah teknik kromatografi adalah teknik pemisahan suatu zat yang didasarkan pada perbadaan kecepatan migrasi komponen-komponen yang dipisahkan diantara dua fase, yaitu fase gerak dan fase diam. Istilah kromatografi pertama kali ditemukan oleh Michael Tswatt (1903), seorang ahli botani asal Rusia. Kromatografi sendiri berasal dari bahasa Yunani yang terdiri dari dua kata yaitu, “chromos” yang berarti warna dan “graphos” yang berarti menulis. Jadi kromatografi berarti penulisan dengan warna. Kromatografi sendiri dapat diklasifikasikan lagi menjadi empat teknik yaitu adalah Kromatografi Kertas (KKT), Kromatografi Lapis Tipis (KLT), Kromatografi Gas Cair (KGC), dan Kromatografi Cair Kinerja Tinggi (KCKT). Pada makalah ini akan dibahas mengenai kromatografi lapis tipis (KLT).
Kromatografi lapis tipis adalah salah satu teknik kromatografi yang banyak digunakan karena penggunaanya yang mudah dan cukup murah. Metode ini menggunakan lempeng kaca atau lembaran plastik yang ditutupi penyerap untuk lapisan tipis dan kering bentuk silika gel, alomina, selulosa dan polianida. Untuk menotolkan larutan cuplikan pada lempeng kaca, pada dasarnya dgunakan mikro pipet/ pipa kapiler. Setelah itu, bagian bawah dari lempeng dicelup dalam larutan pengulsi di dalam wadah yang tertutup (chamber) (Rudi, 2010)[1]

1.2 TUJUAN PENULISAN 1. Mengetahui apa itu kromatografi lapis tipis. 2. Mengetahui prinsip kerja dari kromatografi lapis tipis. 3. Mengetahui peran kromatografi lapis tipis dalam bidang kedokteran.

1.3 MANFAAT PENULISAN
Manfaat dari penulisan makalah ini adalah untuk lebih mengetahui kromatografi lapis tipis serta pengaplikasiannya dalam bidang kedokteran.

BAB II
TINJAUAN PUSTAKA 2.1 DEFINISI
Kromatografi adalah teknik untuk memisahkan campuran menjadi komponennya dengan bantuan perbedaan sifat fisik masing-masing komponennya. Alat yang digunakan terdiri atas kolom yang didalamnya diisikan fasa diam atau stasioner (padatan atau cairan). Campuran ditambahkan ke dalam kolom dari ujung satu dan campuran akan bergerak dengan bantuan pengemban yang cocok (fasa mobil). Pemisahan dicapai oleh perbedaan laju turun masing-masing komponen dalam kolom, yang ditentukan oleh kekuatan adsorpsi atau koefisien partisi antara fasa mobil dan fasa diam (stasioner) (Takeuchi, 2009)[2]
Kromatografi Lapis Tipis (KLT) merupakan salah satu metode isolasi yang terjadi berdasarkan perbedaan daya serap (adsorpsi) dan daya partisi serta kelarutan dari komponen-komponen kimia yang akan bergerak mengikuti kepolaran eluen. Oleh karena daya serap adsorben terhadap komponen kimia tidak sama, maka komponen bergerak dengan kecepatan yang berbeda sehingga hal inilah yang menyebabkan pemisahan (Hostettmann et al, 1995)[3]

2.2 TEORI KROMATOGRAFI LAPIS TIPIS
Kromatologi lapis tipis (KLT) merupakan cara pemisahan campuran senyawa menjadi senyawa murni dan mengetahui kuantitasnya yang menggunakan kromatografi, serta merupakan analisis cepat yang memerlukan bahan sangat sedikit, baik menyerap maupun merupakan cuplikannya. KLT dapat digunakan untuk memisahkan senyawa-senyawa yang sifatnya hidrofolik seperti lipida-lipida dan hidrokarbon yang sukar dikerjakan dengan kromatorgarfi kertas. KLT juga dapat digunakan untuk mencari kromatografi kolom, identifikasi senyawa secara kromatografi dengan sifat kelarutan senyawa yang dinalisis. Bahan lapis tipis seperti silika gel adalah senyawa yang tidak bereaksi dengan pereaksi-pereaksi yang lebih reaktif seperti asam sulfat (Fessenden, 2003)[4]. Kromatografi Lapis Tipis (KLT) pertama kali dikembangkan oleh Izmailoff dan Schraiber pada tahun 1938. (Gandjar dan Rohman, 2007)[5]
Kromatografi lapis tipis (KLT) adalah suatu teknik yang sederhana dan banyak digunakan. Metode ini menggunakan lempeng kaca atau lembaran plastik yang ditutupi penyerap untuk lapisan tipis dan kering bentuk silika gel, alomina, selulosa dan polianida. Untuk menotolkan larutan cuplikan pada lempeng kaca, pada dasarnya digunakan mikro pipet/ pipa kapiler. Setelah itu, bagian bawah dari lempeng dicelup dalam larutan pengulsi di dalam wadah yang tertutup (chamber) (Rudi, 2010)[1]
KLT merupakan metoda kromatografi cair yang melibatkan dua fasa yaitu fasa diam dan fasa gerak. Fasa geraknya berupa campuran pelarut pengembang dan fasa diamnya dapat berupa serbuk halus yang berfungsi sebagai permukaan penyerap (kromatografi cair-padat) atau berfungsi sebagai penyangga untuk lapisan zat cair (kromatografi cair-cair). Fasa diam pada KLT sering disebut penyerap walaupun berfungsi sebagai penyangga untuk zat cair di dalam sistem kromatografi cair-cair. Hampir segala macam serbuk dapat dipakai sebagai penyerap pada KLT, contohnya silika gel (asam silikat), alumina (aluminium oksida), kiselgur (tanah diatomae) dan selulosa. Silika gel merupakan penyerap paling banyak dipakai dalam KLT (Iskandar, 2007)[6]
KLT merupakan bentuk kromatografi planar, yang fase diamnya berupa lapisan seragam (uniform) dengan ketebalan sekitar 0,1-0,3 mm pada permukaan bidang datar yang didukung oleh lempeng kaca, plat aluminium, atau plat plastik (Gandjar dan Rohman, 2007)[5]. Fasa diam yang digunakan dalam KLT merupakan penyerap yang berukuran kecil dengan diameter partikel antara 10-30 µm. Semakin kecil ukuran rata-rata partikel fasa diam dan semakin sempit kisaran ukuran fasa diam, maka semakin baik kinerja KLT dalam hal efesiensi dan resolusinya. Penyerap yang paling sering digunakan adalah silika dan serbuk selulosa. Mekanisme utama pada KLT adalah partisi dan absorbsi. Lapisan tipis yang digunakan sebagai penyerap terbuat dari silika yang telah dimodifikasi. Pada fasa gerak KLT ini berupa satu pelarut atau lebih yang harus memliki kemurnian yang sangat tinggi karena KLT merupakan suatu teknik pemisahan yang sensitif (Gandjar & Rohman, 2012)[7]. Fase diam untuk kromatografi lapis tipis seringkali juga mengandung substansi yang mana dapat berpendar flour dalam sinar ultra violet. Fase gerak merupakan pelarut atau campuran pelarut yang sesuai.
Pada identifikasi noda atau penampakan noda, jika noda sudah berwarna dapat langsung diperiksa dan ditentukan harga Rf. Rf merupakan nilai dari Jarak relatif f pada pelarut. Harga Rf dihitung sebagai jarak yang ditempuh oleh komponen dibagi dengan jarak tempuh oleh eluen (fase gerak) untuk setiap senyawa berlaku rumus sebagai berikut: Bilangan Rf selalu berupa pecahan dan terletak antara 0,01 dan 0,99 (Harbone, 1996)[8]

Contoh hasil dari kromatografi lapis tipis
Contoh hasil dari kromatografi lapis tipis

Kromatografi Lapis Tipis
Kromatografi Lapis Tipis

2.3 PRINSIP KERJA KROMATOGRAFI LAPIS TIPIS
Pada dasarnya KLT digunakan untuk memisahkan komponen-komponen berdasarkan perbedaan adsorpsi atau partisi oleh fase diam di bawah gerakan pelarut pengembang (Watson, 2010)[9]. KLT sangat mirip dengan kromatografi kertas, terutama pada cara pelaksanaannya. Perbedaan nyata terlihat pada fase diamnya atau media pemisahnya, yakni digunakan lapisan tipis adsorben sebagai pengganti kertas. Pada proses pemisahan dengan kromatografi lapis tipis, terjadi hubungan kesetimbangan antara fase diam dan fasa gerak, dimana ada interaksi antara permukaan fase diam dengan gugus fungsi senyawa organik yang akan diidentifikasi yang telah berinteraksi dengan fasa geraknya. Kesetimbangan ini dipengaruhi oleh 3 faktor, yaitu kepolaran fase diam, kepolaran fase gerak, serta kepolaran dan ukuran molekul.
Bahan ditaruh pada daerah terbatas didekat ujung selembar kertas saring atau lapis tipis, dan suatu pelarut atau lapis tipis oleh kerja kapiler. Pada kondisi yang sesuai setelah beberapa waktu, campuran akan dijumpai telah berpindah dari penotolan tadi dan telah berpisah seluruhnya atau sebagian menjadi komponen-komponennya sebagai zona yang jelas. Zona-zona dalam bentuk noda-noda atau pita-pita dapat ditentukan letaknya dengan penggunaan reagensia kimia yang sesuai dengan kertas itu atau oleh pendarah flour-nitra-violet. Difusi pelarut dan pemisahan yang dihasilkan menjadi noda-noda atau pita-pita. (Svehla, 1979)[10]

2.4 PEMBUATAN PLAT LAPIS TIPIS DAN PENYERAP
Penyerap dituangkan diatas permukaan plat yang kondisi bentuknya baik, biasanya digunakan plat kaca / aluminium. Ukuran yang digunakan tergantung pada jenis dari pemisahan yang akan dilakukan dan jenis dari bejana kromatografi. Hal yang penting yaitu bahwa permukaan dari plat harus rata. Plat -plat kaca / aluminium sebelum dipakai dicuci terlebih dahulu dengan air dan detergent kemudian dikeringkan. Terakhir, dapat dicuci dengan aseton, tetapi hal ini tidak mesti dilakukan. Satu hal yang perlu diperhatikan jangan menyentuh permukaan dari plat yang bersih dengan jari tangan karena bekas jari tangan yang menempel akan merubah tebal dari permukaan penyerap pada plat.
Pembuatan penyerap, bahan penyerap dicampur dengan air sampai menjadi bubur, biasanya dengan perbandingan x gram penyerap dan 2x ml air. Bubur diaduk sampai rata dan dituangkan diatas plat dengan berbagai cara. Tebal lapisan merupakan faktor yang paling penting dalam kromatografi lapisan tipis. Tebal standard adalah 250 mikron. Lapisan-lapisan yang lebih tebal ( 0.5 mm - 2.0 mm ) digunakan untuk pemisahan-pemisahan yang sifatnya besar, dengan menggunakan penyerap hingga 250 mg untuk plat dengan ukuran 20 x 20 cm. Salah satu kesukaran dengan lapisan tebal ialah adanya tendensi mengelupas bila kering.
Sifat yang paling penting dari penyerap adalah besar partikel bubur penyerap dan homogenitasnya, karena adhesi terhadap plat sangat tergantung pada kedua sifat tersebut. Besarnya partikel yang biasa digunakan adalah 1 – 25 mikron. Partikel yang butirannya sangat kasar tidak akan memberikan hasil yang memuaskan dan salah satu alasan untuk menaikkan hasil pemisahan adalah menggunakan penyerap yang butirannya halus. Sedangkan dalam kolom partikel yang sangat halus akan mengakibatkan aliran pelarut menjadi lambat, pada lapisan tipis butiran yang halus memberikan aliran pelarut yang lebih cepat. Beberapa contoh penyerap yang digunakan untuk pemisahan-pemisahan dalam kromatografi lapisan tipis adalah sebagai berikut (Kealey dan Haines, 2002)[11] :

Zat padat | Digunakan untuk memisahkan | Silika | Asam- asam amino, alkaloid, gula, asam-asam lemak, lipida, minyak esensial, anion, dan kation organic, sterol, terpenoid. | Alumina | Alkaloid, zat warna, fenol, steroid, vitamin-vitamin, karoten, asam-asam amino | Kieselguhr | Gula, oligosakarida, asam- asam lemak, trigliserida, asam -asam amino, steroid. | Bubuk selulosa | Asam-asam amino, alkaloid, nukleotida | Pati | Asam-asam amino | Sephadex | Asam-asam amino, protein |

2.5 KELEBIHAN DAN KEKURANGAN KROMATOGRAFI LAPIS TIPIS
Kelebihan KLT yaitu: 1. KLT lebih banyak digunakan untuk tujuan analisis. 2. Identifikasi pemisahan komponen dapat dilakukan dengan pereaksi warna, fluoresensi, atau dengan radiasi menggunakan sinar ultraviolet. 3. Dapat dilakukan elusi secara mekanik (ascending), menurun (descending), atau dengan cara elusi 2 dimensi. 4. Ketepatan penentuan kadar akan lebih baik karena komponen yang akan ditentukan merupakan bercak yang tidak bergerak. 5. Hanya membutuhkan sedikit pelarut. 6. Biaya yang dibutuhkan terjangkau. 7. Jumlah perlengkapan sedikit. 8. Preparasi sample yang mudah. 9. Dapat untuk memisahkan senyawa hidrofobik (lipid dan hidrokarbon) yang dengan metode kertas tidak bisa (Gandjar dan Rohman, 2007)[5]

Kekurangan KLT yaitu: 1. Butuh ketekunan dan kesabaran yang ekstra untuk mendapatkan bercak/noda yang diharapkan. 2. Butuh sistem trial and error untuk menentukan sistem eluen yang cocok. 3. Memerlukan waktu yang cukup lama jika dilakukan secara tidak tekun.

2.6 PERANAN KROMATOGRAFI LAPIS TIPIS DALAM BIDANG KEDOKTERAN Dalam bidang kedokteran, teknik ini sangat bermanfaat terutama dalam menginvestigasi fluida badan seperti air liur. Dari air liur seorang pasien, dokter dapat mengetahui jenis penyakit yang diderita pasien tersebut. Seorang perokok dapat diketahui apakah dia termasuk perokok berat atau ringan dengan mengetahui konsentrasi sianida dari sampel air liurnya. Demikian halnya urin, darah dan fluida badan lainya biasa memberikan data yang akurat dan cepat sehingga keberadaan suatu penyakit dalam tubuh manusia dapat dideteksi secara dini dan cepat. Sekarang ini, deteksi senyawa oksalat dalam air kencing menjadi sangat penting terutama bagi pasien kidney stones (batu ginjal). Banyak metode analisis seperti spektrofotometri, atau lainnya, akan tetapi semua membutuhkan kerja ekstra dan waktu yang cukup lama untuk mendapatkan hasil analitis dibandingkan dengan teknik kromatografi.

BAB III
KESIMPULAN DAN SARAN 4.1 KESIMPULAN
Kromatografi Lapis Tipis (KLT) merupakan salah satu metode isolasi yang terjadi berdasarkan perbedaan daya serap (adsorpsi) dan daya partisi serta kelarutan dari komponen-komponen kimia yang akan bergerak mengikuti kepolaran eluen. Dikarenakan adanya perbedaan adsorpsi tersebut, maka terjadilah pemisahan suatu campuran menjadi komponen-komponen. KLT merupakan salah satu metode paling mudah dan murah serta banyak digunakan. Dalam bidang kedokteran sendiri, KLT digunakan dalam mendiagnosa suatu penyakit dengan cepat seperti batu ginjal melalui urin.

4.2 SARAN
Untuk lebih memahami mengenai kromatografi lapis tipis, diharapkan untuk membaca literature yang berhubungan dengan teknik pemisahan campuran khususnya kromatografi lapis tipis.

DAFTAR PUSTAKA

1. Rudi, L. Penuntun Dasar-Dasar Pemisahan Analitik. Kendari : Universitas Haluoleo. 2010. 2. Takeuchi, Yashito. Kromatografi. Tokyo: Iwanami Shoten Publishers. 2009. 3. Hostettmann K, Hostettmann M, Marston A. Cara Kromatografi Preparatif. Bandung : ITB. 1995. 4. Fessenden. Kimia Organik. Jakarta : Erlangga. 2003. 5. Gandjar, IG dan Rohman, A. Kimia Farmasi Analisis. Yogyakarta : Pustaka Pelajar. 2007. 6. Iskandar, Yusuf. Karakteristik Zat Metabolit Sekunder Dalam Ekstrak Bunga Krisan (Chrysanthemum cinerariaefolium) Sebagai Bahan Pembuatan Biopestisida. Semarang : FMIPA. 2007. 7. Gandjar, IG dan Rohman A. Kimia Farmasi Analitis. Yogyakarta : Penebar Swadaya. 2012. 8. Harbone. Metode Fitokimia Penuntun Cara Modern Menganalisis Tumbuhan. Ed ke-2. Bandung : ITB. 1996. 9. Watson, D. Analisis Farmasi. Jakarta : EGC. 2010 10. Svehla, G. Vogel Buku Teks Analisis Anorganik Kualitatif Makro dan Semimakro. Jakarta: PT. Kalman Media Pusaka. 1979. 11. Kealey D dan Haines PJ. 2002. Instant Notes: Analytical Chemistry. New York : BIOS Scientific Publishers Limited. 2002.

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Rapid Detection of Nicotine

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How to Get Out of Jail

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