Latest revision as of 13:04, 6 October 2016

Caffeine can be detected using Reversed Phase High Pressure Liquid Chromatography with UV Detection. (Chris Knutson, 2015)

In order to accomplish caffeine analysis the HPLC system requires the following:

C18 column
UV Detector Monitoring 275nm
Mobile Phase
- Acetonitrile
- 0.1% Acidified Water

Samples should be filtered through 45 μm syringe filters into HPLC sample vials, capped, labeled, and loaded into the HPLC auto-sampler carousel.

High Pressure Liquid Chromatography Setup (HPLC)

Pump Gradient

	Time	Flow	%A	%B	Curve
1		1.00	0.0	100.0
2	3.00	1.00	0.0	100.0	6
3	15.00	1.00	60.0	40.0	6
4	20.00	1.00	60.0	40.0	6
5	25.00	1.00	0.0	100.0	6

Solvents

Solvent A: Acetonitrile
Solvent B: 0.01% Acidified Water

UV Detection

Dual Wavelength Detection
Channel 1: 275nm
Channel 2: 325nm

HPLC Pump Gradient Flow Settings	HPLC Pump Solvent Settings	HPLC UV Detector General Settings
HPLC UV Detector Channel 1 Settings	HPLC UV Detector Channel 2 Settings

Trial Configuration

The samples can be injected in 10 μl increments and trials can range in length from 15-30 minutes depending upon the complexity of one's sample.

Results

Caffeine should be detected as sharp peaks which elute at approximately 11 minutes when monitoring 275nm on the UV Detector.

The image below is the spectra of a caffeine standard run with the above method.

Caffeine Standard Spectra

@@ Line 1: / Line 1: @@
-HPLC method for caffeine analysis
+Caffeine can be detected using Reversed Phase High Pressure Liquid Chromatography with UV Detection. (Chris Knutson, 2015)
-=High Pressure Liquid Chromatography Setup=
+In order to accomplish caffeine analysis the HPLC system requires the following:
-{|align="center"
+*C18 column
+*UV Detector Monitoring 275nm
+*Mobile Phase
+**Acetonitrile
+**0.1% Acidified Water
+Samples should be filtered through 45 μm syringe filters into HPLC sample vials, capped, labeled, and loaded into the HPLC auto-sampler carousel.
+=High Pressure Liquid Chromatography Setup (HPLC)=
+==Pump Gradient==
+{| class="wikitable"
+||
+||Time
+||Flow
+||%A
+||%B
+||Curve
+|-
+||1
+||
+||1.00
+||0.0
+||100.0
+||
+|-
+||2
+||3.00
+||1.00
+||0.0
+||100.0
+||6
+|-
+||3
+||15.00
+||1.00
+||60.0
+||40.0
+||6
+|-
+||4
+||20.00
+||1.00
+||60.0
+||40.0
+||6
+|-
+||5
+||25.00
+||1.00
+||0.0
+||100.0
+||6
+|}
+==Solvents==
+*Solvent A: Acetonitrile
+*Solvent B: 0.01% Acidified Water
+==UV Detection==
+*Dual Wavelength Detection
+*Channel 1: 275nm
+*Channel 2: 325nm
+{|
 ||[[File:1.PNG|thumb|250px|frame|center|HPLC Pump Gradient Flow Settings]]
 ||[[File:2.PNG|thumb|250px|frame|center|HPLC Pump Solvent Settings]]
@@ Line 13: / Line 83: @@
 |}
+=Trial Configuration=
+The samples can be injected in 10 μl increments and trials can range in length from 15-30 minutes depending upon the complexity of one's sample.
 =Results=
-Caffeine should be detected as sharp peaks at 275nm as can be observed in the image below.
+Caffeine should be detected as sharp peaks which elute at approximately 11 minutes when monitoring 275nm on the UV Detector.
-[[File:Std.PNG|thumb|250px|frame|center|Caffeine Standard Spectra]]
+The image below is the spectra of a caffeine standard run with the above method.
+[[File:Std.PNG|thumb|250px|frame|left|Caffeine Standard Spectra]]

Difference between revisions of "Caffeine HPLC Method"