GC Column and Mode operation

temperature controlled oven, door opened to show column from injector exit to detector Kolom dala GC merupaka salah satu komponen utama dalam pemisahan. Kolom berada setelah injektor dan sebelum detektor, dimana terjadi proses pemisahan. Kolom terdapata di dalam oven dengan suhu yang bisa diatur. Besar suhu di dalam kolom ini diatur sedemikian rupa sehingga wujud fasa dalam kolom tetap sesuai dengan ang diinginkan capillary column temperature controlled oven, door opened to show column

Types of GC Column Packed Column Capillary Column Secra umum, didalam GC ada dua jenis kolom yaitu kolom packed dan kapiler

Packed Column stainless steel, nickel, glass, plastics, aluminium. Internal diameter 1.6 to 9.5 mm Length is often 1-3 m Limited Resolution (N< 8,000) Internal column diameter should be at least eight times the diameter of the support particles Densely packed with packing materials or solid support coated with thin layer of stationary liquid phase Diatomaceous earth Size: 60-80 mesh (250-170 m) or 80-100 mesh (170-149 m) Kolom packed merupakan kolom yang biasanya terbuat dari stanless, nikel, kaca, plastik dan aluminium dengan dimeter 1,6 sampai 9 mm dan panjang 1-3 M. Kolom ini memiliki resolusi yang tidak terlalu besar, dimana didalamnya sp alan berada dalam pacekd dengan supproting material, yang biasany a terbuat dai tanah diatomae ( nama dagang: diatoport, celite atao chromosorb,komposisis: silika, al2o3, fe2o3, cao, dll). Chromosorb ini telah dinonaktifkan sehingga mencegah kemungkinan reaksinya dnegan Sp atau MP, dengan melindungi gugus hidroksil dari pendukung, yang sering dipakai dimetilklorosilan dan heksamet

Packed Column Pada packed colomun, Sp memenuhi rongga dari kolom, dan melapisi supporting material.

Capillary Column Internal diameter 1 mm or less. typically <500 μm Usually constructed of fused silica, metal, plastic, glass (fragile) Two kinds of capillarly columns are: packed columns with solid particles over the hole diameter of the column (micropacked) and open tubular columns with an open and unrestricted flow Kolom kapiler biasanya diameternya lbih kecil, 1 mm atau kuran.ada dua macam: packed dengan solid artikel diseluruh diameter kolom atau open tubular

Types of Capillary column Solid support coated with liquid phase Porous Adsorbent Liquid phase Wall coated dibuat dengan memasukkan fasa diam ke dalam kolom sampai terbentuk suatu lapisan yang setimbang pada dinding kolom atau dengan measukkan suatu plug yang rapi ke dalam kolom dengan tekanan, sehingga didapatkan lapisan pada dinding yangs eragam. SCOT dibuat dengan kapastas besar, dimana dengan mendepositkan suatu lapisan tipis diatomite yang sudah dilapisi oleh fasa diam, untuk memberikan suatu ruangan yang luas untuk proses pemisahan. Metode ini sekarang sudah digantikan oleh wcot.fasa diamyang melapisi supporting material mudah menhabiskan kolom dan rusak oleh pelarut dan kontaminan. PLOT dengan alumnia, molekular sieve dan coplymer surface coating untuk pemisana alkana C1-C6, alkena dan gas atmosfer yang memiliki retensi rendah, yang ditingkatkan dengan mengganti lapisan film fasa diam dengan adsorbent fasa diam untuk meningkatkan mekanisme retensi Wall-coated Open Tubular (WCOT) Support-coated Open Tubular (SCOT) Porous Layer Open Tubular (PLOT) FSOT: Fused-silica open tubular column

Characteristics Type of Column PLOT WCOT SCOT Packed Length (m) 10-100 1-6 ID (mm) 0.1-0.3, 0.53* 0.25-0.75 0.5 2-4 Efficiency (Plate/m) 2000-4000 1000-4000 600-1200 500-1000 Sample size (ng) 10-75 10-1000 10-106 Relative pressure Low High Relative speed Fast Slow Flexible Yes No Chemical inertness Best Poor *Megabore column

For capillary GC columns…. Increased length = greater N, therefore a greater R expense is possible band broadening if analytes are on the column too long! Increased length leads to longer separations. Do you have the time? Increased stationary phase thickness and column diameter provides increased sample capacity and can provide increased resolution tradeoffs are a longer analysis time and more column bleed with thicker stationary phases Is the column compatible with the detector? Thick stationary phases bleed more and will contaminate a mass spectrometer. For most analytical work, a best “compromise” column is chosen and other variables (temp, etc.) are altered to optimize the separation. Dalam kolom kapiler, kenaikan anjang kolom jug meningkatkan nilai N, sehingga nilai resolusi juga akan naik.kelemhahan: kemungkinan besar ada pelebaran puncak jika analit terlalu lama dalam kolom dan waktu analisis lama Kenaikan ketebalan fasa diam dan diameter kolom -- kenaikan kapasitas dan kenaikan resolusi . Kelemahan: semakin tebal, smakin lama dan kemungkinan kolom bleeding

Stationary Phases Low volatility, thermal stability, chemical inertness Provide k and  within a suitable range consider the polar characteristics of the analytes and select SP of similar polarity ‘Like dissolves like’

Column Dimensions Column Length: 10 – 60 m Column Internal Diameter: 0.10 – 0.53 mm Stationary Phase Film Thickness: 0.10 – 0.25 mm

Stationary Phases Solid phase Liquid phase Most uses for separation of low MW compounds and gases Common SP: silica, alumina, molecular sieves such as zeolites, cabosieves, carbon blacks Liquid phase Over 300 different phases are widely available grouped liquid phases Non-polar, polar, intermediate and special phases Polymer liquid phase

Stationary Phase Polymers Siloxane Arylene Polyethylene glycol

Liquid phases Non-polar phase Polar phase Intermediate phase Primarily separated according to their volatilities Elution order varies as the boiling points of analytes Common phases: dimethylpolysiloxane, dimethylphenylpolysiloxane Polar phase Contain polar functional groups Separation based on their volatilities and polar-polar interaction Common phases: polyethyleneglycol Intermediate phase

Bonded and Cross-linked SP Polymer chains Cross-linking Bonding Fused silica tubing surface Bonded and cross-linked SP provides long term stability, better reproducibility and performance.

Common stationary phase coating for capillary column Composition Polarity Applicaitons Temp limits 100% dimethyl polysiloxane (Gum) Nonpolar Phenols, Hydrocarbons, Amines, Sulfur compounds, Pesticides, PCBs -60oC to 325oC 100% dimethyl polysiloxane (Fluid) Amino acid derivatives, Essential oils 0oC to 280oC 5% diphenyl 95% dimethyl polysiloxane Fatty acids, Methyl esters, Alkaloids, Drugs, Halogenated compounds 14% cyanopropyl phenyl polysiloxane Immediate Drugs, Steroids, Pesticides -20oC to 280oC 50% phenyl, 50% methyl polysiloxane Drugs, Steroids, Pesticides, Glycols 60oC to 240oC 50% cyanopropylmethyl, 50% phenylmethyl polysiloxane Fatty acids, Methyl esters, Alditol acetates 50% trifluoropropyl polysiloxane Halogenated compounds, +Aromatics 45oC to 240oC Polyethylene glycol – TPA modified Polar Acids, Alcohols, Aldehydes acrylates, Nitriles, Ketones Polyethylene glycol Free acids, Alcohols, Ethers, Essential oils, Glycols, Solvents 60oC to 220oC

Stationary Phase CHEMICALLY STABIL AND INERT DISPERSED ON THE SOLID SUPPORT LOW VISCOSITY (FOR FAST MASS TRANSFER) SOLUBLE IN ORGANIC SOLVENTS THERMALLY STABIL LOW VAPOUR PRESSURE HIGH SELECTIVITY FOR COMPOPUNDS OF INTEREST

Stationary Phase

Comparison between packed and WCOT

Choosing a GC Column… Is the column compatible with your analytes polar analytes require polar stationary phases so they will spend some of their “time” in the stationary phase non-polar analytes require non-polar stationary phases You usually have to compromise on the stationary phase to get a good column for your analytes (which are probably a mix of polar and non-polar) DB-5, HP-5, EC-5, RTX-5 (5% dimethyl, 95% diphenyl polysiloxane) most common general use column. Temperature range, solvent and carrier gas compatibility Sample capacity versus resolution usually determines packed vs.. capillary GC’s usually setup for either packed or capillary Let’s say you choose a capillary column, there’s more to think about!

Chromatogram

Column performance B A B A Concentration Broadening Distance migrated

If the length of the column is L, then the HETP is column efficiency Theoretical Plate Model of Chromatography theoretical plates is a number of a separate layers, where separate equilibrations of the sample between the stationary and mobile phase occur in Plates are a figment of the imagination that helps us understand the processes at work in the column.They also serve as a way of measuring column efficiency, either by stating the number of theoretical plates in a column, N (the more plates the better), or by stating the plate height; the Height Equivalent to a Theoretical Plate (the smaller the better). If the length of the column is L, then the HETP is HETP = L / N

Van Deemter equation for plate height; The number of theoretical plates that a real column possesses can be found by examining a chromatographic peak after elution; where w1/2 is the peak width at half-height Van Deemter equation for plate height; HETP = A + B / u + C u where u is the average velocity of the mobile phase. A, B, and C are factors which contribute to band broadening

A - Eddy diffusion The mobile phase moves through the column which is packed with stationary phase. Solute molecules will take different paths through the stationary phase at random. This will cause broadening of the solute band, because different paths are of different lengths. B - Longitudinal diffusion The concentration of analyte is less at the edges of the band than at the center. Analyte diffuses out from the center to the edges. This causes band broadening. If the velocity of the mobile phase is high then the analyte spends less time on the column, which decreases the effects of longitudinal diffusion. C - Resistance to mass transfer The analyte takes a certain amount of time to equilibrate between the stationary and mobile phase. If the velocity of the mobile phase is high, and the analyte has a strong affinity for the stationary phase, then the analyte in the mobile phase will move ahead of the analyte in the stationary phase. The band of analyte is broadened. The higher the velocity of mobile phase, the worse the broadening becomes.

LONGITUDINAL DIFFUSION

MASS TRANSPORT

RESOLUTION Another measure of how well species have been separated is provided by measurement of the resolution. The resolution of two species, A and B, is defined as Baseline resolution is achieved when R = 1.5

EFFICIENCY SEPARATION

EFFICIENCY OF SEPARATION: PEAK RESOLUTION

GAUSSIAN PEAKS

PEAK RESOLUTION

Column Preparation A known weight of support is mixed with 3 to 4 times its volume of a volatile solvent containing a weight of the support particles. the solvent is then evaporated, often in a rotary stripper. To give a dry, free flowing solid, which is slowly poured in to the column with gentle trapping

Temperature Programming in GC The “simplest” way to alter the separation in GC is to alter the temperature program in the oven. You can also alter the pressure of the carrier gas, but this is less common (much). Isothermal = constant temperature Gradient = varied temperature By altering the temperature, you vary the rate of the reaction for any analyte: they spend more or less time in the stationary phase the greater the difference in the times between analytes, the better the separation!

The traps of temperature… If your temperature at a given time is too high, you can cause the peaks to co-elute poor resolution vs but a faster separation If your temperature at a given time is too low, you can get still get a good separation adequate resolution, but a separation that takes very long You have to choose a compromise temperature program

Temperature program oven or run isothermally

Control Panel sets Separation Method

Column Temperature Diatas titik lebur liquid phase, dibawah suhu maksimum yang diperbolehkan dari fasa cair Suhu kolom berdasarkan kompromi, suhu rendah, pemisahan lebih baik, waktu retensi lama. Kompromi= rata-rata titik didih cuplikan Penurunan suhu kolom 30˚c, waktu retensi 2 kali lipat

Soal 1 Dapatkah saudara meramalkan urutan elusi pada apiezon, OV-17 dan carbowax dari senyawa-senyawa dalam cuplikan berikut: 1-butil bromida Dietil keton Metil sikloheksana

Jawab Apiezon: dietil keton-> 1-butil bromida -> metil sikloheksana (kenapa 1-butil bromida keluar setelah dietil keton?) OV-17: metil sikloheksana -> dietil keton -> 1-butil bromida Carbowax: metil sikloheksana -> 1-butil bromida -> dietil keton (kenapa dietil keton keluar lebih lama dibandingkan 1-butil bromida?)

Soal 2 Urutan elusi propanol, 1-propil asetat dan 1-heptana pada kolom apiezon, ov-17 dan carbowax

Jawab Apiezon: propanol -> 1-propil asetat -> 1-heptana OV-17: propanol -> 1-heptana -> 1-propil asetat (kenapa 1-heptana keluar lebih dulu daripada 1-propil asetat?) Carbowax: 1-heptana -> 1-propil asetat -> propanol

Soal 3 Mengapa resolusi yang baik dengan nilai Rs minimal 1,5? Mengapa dalam GC tekanan sangat penting dan tekanan gas pembawa harus konstan?