136 Draft. May Mga Kulang Pa

COLUMN CHROMATOGRAPHY:
HISTORY and APPLICATIONS

MIKHAIL TWSETT Year 1903
Separate plant pigments to write with colors method of separating mixtures based on the compounds affinity to the stationary phase and the mobile phase separation technique a sample preparation method to isolate and purify the target compound for further analyses
Now and Then
20th Century
Separation of Drugs
As a separation technique
Or as a sample preparation technique
Separation of Drugs
MOBILE PHASE
STATIONARY PHASE
1:1 hexane/diethyl ether 1:2 hexane dietheyl ether acetone
silica
Separation of Drugs ASPIRIN
COOH O O CH 3
PARACETAMOL
HO O N H CH 3
CAFFEINE
O H 3C N N CH3 CH 3 N
Other Names: 2acetoxybenzoic acid; acetylsalicylic acid Solubility: 1 g dissolves in 300 ml water at 25, in 100 ml water at 37, in 5 ml alcohol Absorption maximum: uv max (0.1N H2SO4): 229 nm (E1%1cm 484); (CHCl3): 277 nm (E1%1cm 68) Therap-Cat: Analgesic; antipyretic; antiinflammatory.
Other Names: N-(4Hydroxyphenyl)acetamide; paracetamol Solubility: Very slightly sol in cold water, considerably more sol in hot water. Sol in methanol and ethanol. Absorption maximum: uv max (ethanol): 250 nm (e 13800) Therap-Cat: Analgesic; antipyretic.
Other Names 1,3,7trimethylxanthine Solubility: 1 g dissolves in 46 ml water, 5.5 ml water at 80, 1.5 ml boiling water, 66 ml alcohol, 22 ml alcohol at 60. Absorption maximum: uv max (water at 25ug/mL): 205 nm; 273 nm. Therap-Cat: CNS stimulant; respiratory stimulant.
Separation of Drugs
SILICA
Slightly acidic medium Very polar compound Best for ordinary compounds
Separation of Drugs
COLUMN EFFICIENCY
Plate Height Plate Number
VARIABLES: Diffusion coefficient of the stationary phase Diameter of the packing particles Diffusion coefficient in the mobile phase Thickness/Height of the packing Retention factor Linear velocity of the mobile phase
= GOOD SEPARATION
OBJECTIVES
To optimize the column chromatographic separation of aspirin, paracetamol, and caffeine with silica as the stationary phase and using isocratic elution by:
determining the most efficient solvent system to be used as the mobile phase determining the most efficient size of the column.
METHODS
THIN LAYER CHROMATOGRAPHY

Sample Preparation Stationary Phase
Mixed solution: 2.5 mg/mL each of ASA, PCT and CAF
(3 replicates)
Silica gel GF254 precoated plate
Mobile Phase
Ethyl acetate 4:1 ethyl acetate: hexane UV at -nm
Solvent Front Detection Method
COLUMN CHROMATOGRAPHY CONDITIONS
STATIONARY PHASE
MOBILE PHASE
FLOW RATE
PARAMETERS OF THE COLUMN
STATIONARY PHASE SILICA
MOBILE PHASE
FLOW RATE
Conditions of TLC
STATIONARY PHASE SILICA
MOBILE PHASE
FLOW RATE
Conditions of Column Chrom Packing of Column, etc.
RESULTS
THIN LAYER CHROMATOGRAPHY

[Mobile Phase] [Mobile Phase]
Whats the most appropriate Mobile Phase?
COLUMN CHROMATOGRAPHY
2.75 2.25 Absorbance
Column: 10 mL
ABSORBANCE vs FRACTION (per max): Total Absorbance
230 nm 244 nm
1.75
1.25 0.75 0.25 -0.25
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Were the drugs separated?
274 nm
18
19
20
Fraction
Column: 5 mL
230 nm 244 nm
1.75
1.25 0.75 0.25 -0.25
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
274 nm
19
20
21
Fraction
Column: 3 mL
230 nm 244 nm
1.75
1.25 0.75 0.25 -0.25
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
274 nm
19
20
21
Fraction
Beer-Lamberts Law Brief summary Focus on direct relationship between absorbance and concentration
MATRIX RESOLUTION METHOD CALCULATING CONC OF EACH DRUG
MATRIX RESOLUTION METHOD SCANNING VALUES USED TO CALC MOLAR ABSORPTIVITY
3.50 3.00 2.50 2.00
Column: 10 mL max: 230 nm
ABSORBANCE vs FRACTION (per max): INDIVIDUAL Absorbance

ASA
PCT
CAF
Absorbance
1.50 1.00 0.50 0.00 -0.50 -1.00
-1.50
-2.00
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Fraction
3.50 3.00 2.50 2.00

ASA
PCT
CAF
Absorbance
1.50 1.00 0.50 0.00 -0.50 -1.00
-1.50
-2.00
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Fraction
3.50 3.00 2.50 2.00

ASA
PCT
CAF
Absorbance
1.50 1.00 0.50 0.00 -0.50 -1.00
-1.50
-2.00
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Fraction
3.50 3.00 2.50 2.00

ASA
PCT
CAF
Absorbance
1.50 1.00 0.50 0.00 -0.50 -1.00
-1.50
-2.00
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Fraction
3.50 3.00 2.50 2.00

ASA
PCT
CAF
Absorbance
1.50 1.00 0.50 0.00 -0.50 -1.00
-1.50
-2.00
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Fraction
3.50 3.00 2.50 2.00

ASA
PCT
CAF
Absorbance
1.50 1.00 0.50 0.00 -0.50 -1.00
-1.50
-2.00
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Fraction
3.50 3.00 2.50 2.00

ASA
PCT
CAF
Absorbance
1.50 1.00 0.50 0.00 -0.50 -1.00
-1.50
-2.00
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Fraction
3.50 3.00 2.50 2.00

ASA
PCT
CAF
Absorbance
1.50 1.00 0.50 0.00 -0.50 -1.00
-1.50
-2.00
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Fraction
3.50 3.00 2.50 2.00

ASA
PCT
CAF
Absorbance
1.50 1.00 0.50 0.00 -0.50 -1.00
-1.50
-2.00
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Fraction
Calculation of Parameters for Column Efficiency
From the results, it can be said that ______ is the most efficient column. > insert again here the calculated parameters. Describe interaction of the drugs to the stationary phase and column. Kung bakit si Caffeine ay laging nauuna, etc. Reason for negative values.

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COLUMN CHROMATOGRAPHY:

HISTORY and APPLICATIONS

Now and Then

Or as a sample preparation technique

1:1 hexane/diethyl ether 1:2 hexane dietheyl ether acetone

THIN LAYER CHROMATOGRAPHY

Silica gel GF254 precoated plate

Ethyl acetate 4:1 ethyl acetate: hexane UV at -nm

Solvent Front Detection Method

COLUMN CHROMATOGRAPHY CONDITIONS

PARAMETERS OF THE COLUMN

COLUMN CHROMATOGRAPHY CONDITIONS

STATIONARY PHASE SILICA

PARAMETERS OF THE COLUMN

COLUMN CHROMATOGRAPHY CONDITIONS

STATIONARY PHASE SILICA

PARAMETERS OF THE COLUMN

Conditions of Column Chrom Packing of Column, etc.

THIN LAYER CHROMATOGRAPHY

Whats the most appropriate Mobile Phase?

ABSORBANCE vs FRACTION (per max): Total Absorbance

Were the drugs separated?

ABSORBANCE vs FRACTION (per max): Total Absorbance

Were the drugs separated?

ABSORBANCE vs FRACTION (per max): Total Absorbance

Were the drugs separated?

Column: 10 mL max: 230 nm

ABSORBANCE vs FRACTION (per max): INDIVIDUAL Absorbance

1.50 1.00 0.50 0.00 -0.50 -1.00

Were the drugs separated?

Column: 10 mL max: 244 nm

ABSORBANCE vs FRACTION (per max): INDIVIDUAL Absorbance

1.50 1.00 0.50 0.00 -0.50 -1.00

Were the drugs separated?

Column: 10 mL max: 274 nm

ABSORBANCE vs FRACTION (per max): INDIVIDUAL Absorbance

1.50 1.00 0.50 0.00 -0.50 -1.00

Were the drugs separated?

Column: 5 mL max: 230 nm

ABSORBANCE vs FRACTION (per max): INDIVIDUAL Absorbance

1.50 1.00 0.50 0.00 -0.50 -1.00

Were the drugs separated?

Column: 5 mL max: 244 nm

ABSORBANCE vs FRACTION (per max): INDIVIDUAL Absorbance

1.50 1.00 0.50 0.00 -0.50 -1.00

Were the drugs separated?

Column: 5 mL max: 274 nm

ABSORBANCE vs FRACTION (per max): INDIVIDUAL Absorbance

1.50 1.00 0.50 0.00 -0.50 -1.00

Were the drugs separated?

Column: 3 mL max: 230 nm

ABSORBANCE vs FRACTION (per max): INDIVIDUAL Absorbance

1.50 1.00 0.50 0.00 -0.50 -1.00

Were the drugs separated?

Column: 3 mL max: 244 nm

ABSORBANCE vs FRACTION (per max): INDIVIDUAL Absorbance

1.50 1.00 0.50 0.00 -0.50 -1.00

Were the drugs separated?

Column: 3 mL max: 274 nm

ABSORBANCE vs FRACTION (per max): INDIVIDUAL Absorbance

1.50 1.00 0.50 0.00 -0.50 -1.00

Were the drugs separated?

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