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Liquid Chromatograpy – mass spectroscopy adalah dua  alat yang digabungkan menjadi satu, dimana berfungsi untuk memisahkan beberapa senyawa atau campuran senyawa berdasarkan kepolarannya (prinsip kerja kromatograpi), dimana setelah campuran senyawa tersebut terpisah, maka senyawa yang murni tersebut akan diidentifikasi berat moleculnya. Berbeda dengan Gas Chromatograpy – mass spectroscopy, LC-MS pada output datanya sangat jarang sekali terjadi pola fragmentasi, dikarenakan tidak ada proses fragmentasi. Sehingga yang didapatkan adalah berat molekul ditambah beberapa muatan ditambah lagi berat molekul pelarut (terkadang).

ESI adalah salah satu methode untuk mendapatkan berat molekul yang digunakan dalam LC-MS dimana jika pada metode biasanya (lupa-red) menggunakan fragmentasi (pemecahan molekul), maka pada metode ESI menggunakan spray (Penyemprotan). AKibatnya tidak akan ditemukan fragmen –fragmen dari molekul tersebut.

Adapun cara kerja liquid chromatograpi adalah sama dengan HPLC atau liquid chromatograpy lain pada umumnya. Sedangkan kerja Mass spectroscopy metode ESI  adalah sebagai berikut : 

image 

Gambar 1. Proses pemisahan analite pada Liquid Chromatograpy sampai dengan sprayer (penyemprotan oleh spray needle tip)

1. Analyte bersama dengan eluent dari syringe pump atau LC masuk ke dalam cappilary.

Di dalam kappilary terdapat anoda (kutup negatif) pada taylor cone dan katoda (kutup negatif) didekat masukkan analyte dan eluent. Kutup ini berfungsi agar muatan yang berkumpul pada taylor cone adalah muatan positif sehingga nantinya saat terjadi penyemprotan dan terbentuk droplet (tetes – tetes) tidak bergabung – gabung menjadi droplet yang lebih besar lagi.

2.  Analylet dan solven(eluent) di semprotkan (spray) melalui taylor cone.

Akan terbentuk droplet – droplet dimana droplet – droplet itu akan mengalami tahap evaporasi solven untuk mengurangi solven yang menempel di analyte. Karena suatu saat , apabila terjadi evaporasi secara terus menerus maka solven yang meliputi analyte terkungkung dalam muatan positif yang berlebih, dalam bahasa inggris tahap seperti ini disebut the ‘rayleigh’  limit is reached, maka akan terjadi explosion yang disebut coulombic explosion dimana akan terjadi pemecahan droplet (tetesan) tadi. Ada beberapa kemungkinan yang terjadi pada droplet – droplet tersebut .

a. Yang pertama analyte akan tertambahi satu muatan positif

b. yang kedua analyte akan tertambahi beberapa muatan positif

c. yang ketiga analyte akan tertambahi satu muatan positif dan satu molekul solven

d. yang keempat analyte akan tertambahi satu muatan positif dan beberapa molekul solven

e. yang kelima analyte akant tertambahi beberapa muatan positif dan beberapa molekul solven.

image

Gambar 3.Proses Setelah analite dipisahkan oleh Liquid Cromatography

3. Droplet yang mengalami coulombic exsploison tersebut akan masuk ke dalam cone dimana di sisi kiri dan kanannya sudah mengalir gas Nitrogen (N2). Gas ini berfungsi agar analyte yang terjadi tadi stabil dalam bentuknya dan tidak terganggu oleh pengaruh gas oksigen. Droplet masuk ke dalam cappilary transfer lalu akan di analisis melalui mass spectrometer.

image

Gambar 3. Proses Droplet berubah menjadi analyte yang siap untuk di mass spectroscopy

image

gambar 4. Penjelasan datangnya muatan positif pada droplet – droplet LC-MS

image

Gambar 5. Penjelasan kenapa pada LC-MS solven harus di buat muatan positif

 

image gambar 6. Penjelasan kenapa bukan ion negatif yang digunakan

image gambar 7. Penjelasan ion molekul semu apa saja yang terjadi /yang bisa muncul pada output data LC-MS

image

gambar8. Contoh spektra dan hasil

image

image

Gambar 9. Kerugian yang dihasilkan dibandingkan dengan LC-MS

image

Gambar 10. Kesimpulan , diambil dari presented Jessica & Gilement, 2002.

DAFTAR PUSTAKA

  • Adamovics, & John.A, 1997, Chromatographic Analysis of Pharmaceutical, Marcel dekker Inc, New York.

  • Ardrey, R. E., 2003, Liquid chromatography-mass spectrometry: an introduction, 185, John Wiley & Sons, New York.

  • De Hoffmann, E., & Stroobant, V.,2007, Mass spectrometry: principles and applications, 226-227, Wiley-Interscience, New York.

  • Jesicca & Gilment, 2002, Electrospray ionization Mass Spectrometry, Presented at 17 september 2002.

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