HPLC

HPLC or high performance liquid chromatography is a technique for separating components in a sample on the basis of interactions between the component, a liquid mobile phase and a solid stationary phase. Both individual compounds and more complex fractions of a sample can be separated in this manner with detection possible using UV absorption, refractive index measurement, evaporative light scattering or mass spectrometry. The sample is introduced into a metal column coated with a stationary phase through which the mobile phase flows under pressure. The stronger the interaction with the stationary phase, the longer the component will be retained on the column and the stronger the interaction with the mobile phase the shorter will be its residence time on the column and vice versa. A typical separation may require 5 minutes to an hour. HPLC has found its greatest applicability in the pharmaceutical and biotechnology areas in the last 20 years due to its ability to separate complex molecules like proteins and drugs.

Contents

  • 6-B-Hydroxymethandrostenolone & methandrostenolone by HPLC/ELSD
    Anabolic Steroids- 6-B-Hydroxymethandrostenolone and methandrostenolone, Alltech Application Note 0034E, May 14, 2003. The anabolic steroids B-Hydroxymethandrostenolone and methandrostenolone were separated by HPLC/ELSD. Separation of the anabolic steroids B-Hydroxymethandrostenolone and methandrostenolone was achieved by reversed phase isocratic elution with acetonitile/water in around 6 minutes using an Adsorbosphere C8, 5m, 250 x 4.6mm column (Part No. 287112). Detection was by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector). In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Acetone as Solvent for Carbohydrates by HPLC/ELSD
    Alternative Solvents for Carbohydrate Analyses by HPLC, Alltech Application Note 0054E, March 29, 2004. The use of acetone in the mobile phase to speed up run times for carbohydrates in beer by reversed phase HPLC and evaporative light scattering detection is described. Seven sugars and oligomers in a beer standard (fructose, glucose, sucrose, maltose, maltotriose, maltotetraose and maltopentose) were separated by gradient elution with acetonitrile/water and with acetonitrile/acetone and water on a Prevail Carbohydrate ES, 5m, 250 x 4.6mm column (Part No. 35101) and detected by evaporative light scattering using an Alltech 2000 ELSD (Evaporative Light Scattering Detector.) The separation using the unmodified mobile phase took 35 minutes, while that using the acetone took only 18. Likewise, a mixture of fructose, mannitol, glucose, sucrose, raffinose and stachyose was separated in 16 minutes using the acetone modifier and a similar elution gradient. In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Amino Acids L-Histidine Standard by HPLC/ELSD, UV
    Amino Acids L-Histidine Standard, Alltech Application Note 0011E, June 23, 2000. A 99% pure L-histidine amino acid standard was separated from its impurities isocratically in around 5 minutes by reversed phase HPLC on an Absorbosphere C18, 5m, 250 x 2.1mm column (Part No. 9945) using methanol/water and was detected by UV at 254nm and by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector.) The UV detection did not resolve the L-histidine from the impurities, while the ELSD produced distinct peaks for L-histidine and the impurities. In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Beer Oligomers by HPLC/ELSD
    Carbohydrate Profiles-Beer, Alltech Application Note 0055E, January 18, 2002. Oligosaccharides found in beer were separated by gradient elution in around 45 minutes by reversed phase HPLC on a Prevail Carbohydrate ES, 5m, 250 x 4.6mm column (Part No. 35101) using acetonitrile/water and detected by evaporative light scattering using an Alltech 2000 ELSD (Evaporative Light Scattering Detector). Frucotose, glucose, maltose, maltotriose, maltopentaose and maltohexaose were separated in New Castle Ale and maltotriose, maltohexaose and maltoheptaose were separated in Goose Island Beer. In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Caffeine and Aspirin by HPLC/ELSD
    Pharmaceutical-Caffeine and Aspirin, Alltech Application Note 0028LE, June 23, 2000. Caffeine and aspirin were separated by HPLC/ELSD. Separation of caffeine and aspirin was achieved by reversed phase isocratic elution with water/methanol/acetic acid in around 2 minutes using an Econosphere C18, 3m, 30 x 4.6mm column (Part No. 28213). Detection was by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector). In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Carbohydrates - Sugar Standards by HPLC/ELSD
    Carbohydrates Sugar Standards by HPLC/ELSD, Alltech Application Note 0004E, October 23, 2000. A mixture of six sugars were separated in around 26 minutes by reversed phase HPLC on an Adsorbosphere NH2, 250 x 4.6 mm column (Part No. 287242) using both isocratic and gradient elution with acetonitrile/water and detected by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector.) Fructose, glucose, sucrose, maltose and lactose were detected by both forms of elution, while raffinose was detected using gradient only. In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Carbohydrates in Fruit Juices by HPLC/ELSD
    Carbohydrates Fruit Juices, Alltech Application Note 0012E, May 14, 2003. Mixture of sugars found in fruit juices were separated isocratically in around 25 minutes by reversed phase HPLC on a Prevail Carbohydrate ES, 5m, 250 x 4.6mm column (Part No. 35101) using acetonitrile/methanol/water. They were detected by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector). By varying the mobile phase composition, fructose, glucose, sucrose, maltose and lactose were separated in one elution and fructose, glucose, sucrose and raffinose in another elution. In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Carbohydrates in Infant Formula by HPLC/ELSD
    Analysis of Carbohydrates in Soy Infant Formula, Alltech Application Note 0044E, March 29, 2004. Carbohydrate sugars in soy infant formula were determined by reversed phase HPLC with evaporative light scattering detection. The fats in the infant formula were first extracted using hexane and other cleanup procedures performed prior to the HPLC analysis. Glucose, sucrose, maltose and raffinose were separated isocratically and by gradient elution with acetonitrile/water on an Alltima Amino, 5m, 250 x 4.6mm column (Part No. 88217) and detected by evaporative light scattering using an Alltech MKIII ELSD (Evaporative Light Scattering Detector.) The isocratic elution took 16 minutes while the gradient elution took around 12. The ELSD was found to be more sensitive than refractive index detection and its response linear for these sugars. In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Chiral Separation of d and l Propanolol HCl by HPLC/ELSD
    Chiral Separation of d and l Propanolol Hydrochloride, Alltech Application Note 0031E, June 23, 2000. The chiral separation of d and l forms of propanolol hydrochloride was performed by reversed phase HPLC using isocratic elution in around 20 minutes on an Astec Cyclobond 1 2000, 5m, 250 x 4.6mm column (Part No. 410101) using TEA and acetonitrile/methanol. Detection was achieved both UV at 254 nm and by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector). In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Chiral Separation of Warfarin Enantiomers by HPLC/ELSD
    Chiral Separation of Warfarin Enantiomers, Alltech Application Note 0032E, May 2, 2000. The chiral separation of d and l forms of warfarin was performed by reversed phase HPLC using isocratic elution in around 6 minutes on an Astec Cyclobond 1 2000, 5m, 250 x 4.6mm column (Part No. 410101) using acetic acid/TEA and acetonitrile/methanol. Detection was achieved both UV at 254 nm and by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector). In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Cholesterol by HPLC/ELSD
    Cholesterol, Alltech Application Note 0001E, May 8, 2000. A reversed phase HPLC chromatogram of a 104ng/l cholesterol standard is depicted. Cholesterol was separated in 16 min on a Solvent Miser C18, 5m, 250 x 2.1mm column (Part No, 9945) using gradient elution with acetonitrile and methanol. Cholesterol was detected by evaporative light scattering detection (ELSD) using an Alltech 500 ELSD (Evaporative Light Scattering Detector.) In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Combinatorial Sample Analysis by HPLC/ELSD
    Combinatorial Sample Analysis, Alltech Application Note 0050LE, July 7, 2000. Evaporative light scattering detection (ELSD) is presented as an alternative to MS and UV detection from HPLC separations of combinatorial samples, pharmaceutical compounds produced for the first time for which there are no standards. Because the response of the ELSD is based entirely on the mass of the sample, internal standards can be used instead of standards of the target analytes. An n-methyl-d-aspartate antagonist in a drug synthesis solution was separated from its impurities by gradient elution in around 20 minutes by reversed phase HPLC on a Zorbax SB-Phenyl, 250 x 4.6mm column (Agilent Technologies Part No. 880975-912) using water/acetonitrile and detected by evaporative light scattering using an Alltech Model 2000 (Evaporative Light Scattering Detector.) The same separation with UV detection gave misleading results due to weak chromophores for the antagonist and strong ones for the impurities. A mixture of catecholamines (noradrenaline, adrenaline, 3-hydroxytyramine, 3,4-dihydroxyphenylalanine, phenylalanine and homovanillic acid) was separated by gradient elution in around 7 minutes by reversed phase HPLC on a Platinum EPS C18, 3m, 53 x 7mm Rocket column (50573) using water and TFA/acetonitrile and detected by the ELSD. The same separation with UV detection suffered from a drifting baseline. In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Corn Syrup Oligomers by HPLC/ELSD
    Carbohydrate Profiles-Confectionary Sweeteners, Alltech Application Note 0019E, January 18, 2002. Oligosaccharide sweeteners in corn syrup and in commercial maltodextrin were separated isocratically and by gradient elution in around 25 minutes by reversed phase HPLC on a Prevail Carbohydrate ES, 5m, 250 x 4.6mm column (Part No. 35101) using acetonitrile/water and detected by evaporative light scattering using an Alltech 2000 ELSD (Evaporative Light Scattering Detector). Dextrose, maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose and maltooctaose were separated isocratically while these oliogomers and others in the series were separated by gradient elution. These other oligomers were maltononaose, maltodecaose, maltoundecaose, maltododecaose, maltotridecaose, maltotetradecaose and maltopentadecaose. The maltodextrin was separated by gradient elution. Glucose (DPI), DP3 (1,6-alpha; 1,4 alpha), DP6 (1,6-alpha; 1,4 alpha), DP13 and DP18 were identified. In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Cottonseed, Linseed Oil by HPLC/ELSD
    Seed Oils - Cottonseed, Linseed, Alltech Application Note 0002E, May 21, 2000. In separate determinations, cottonseed and linseed oil were separated by reversed phase HPLC on a Solvent Miser C18, 5m, 250 x 2.1mm column (Part No. 9945) using gradient elution with acetonitrile and ethanol. Their respective components were detected by evaporative light scattering detection (ELSD) using an Alltech 500 ELSD (Evaporative Light Scattering Detector.) In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser. Several unidentified components in each oil eluted over a 40-minute period.
  • Detecting Adulteration in Orange Juice by HPLC/ELSD
    Orange Juice Profiles, Alltech Application Note 0052E, January 15, 2002. The ratio of glucose, fructose and sucrose in pure orange juice is well known. Adulteration with high fructose corn syrup is sometimes done. To detect this form of adulteration, an HPLC application with evaporative light scattering detection was developed and described. These sugars in Minute Maid 100% OJ, Dominicks 100% OJ and Dominicks Orange Drink were separated isocratically with acetonitrile/water on a Prevail Carbohydrate ES, 53 x 7mm Rocket column (Part No. 35104) and detected by evaporative light scattering using an Alltech 2000 ELSD (Evaporative Light Scattering Detector.) Fructose, glucose and sucrose in the pure orange juices were all found at about the same ratio, while the levels of fructose and glucose were nearly 3 times higher in the Orange Drink and no sucrose was found. Although the orange drink in this study was not an adulterated product, the application shows how such adulteration could be detected. In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Dimethicone by Reversed Phase HPLC/ELSD
    Dimethicone, Alltech Application Note 0048E, July 7, 2000. Dimethicone, a silicone derived oil, is commonly used in cosmetics, hair care products and pharmaceutical processing. Its analysis is difficult and is usually performed by FT-IR. A dimethicone standard and a sample of hand lotion containing dimethicone were extracted with hydrochloric acid and methylene chloride. The organic phase of the standard was separated in 20 minutes by gradient elution with acetontrile/chloroform on an Alltima C8, 5, 150 x 4.6mm reversed phase HPLC column (Part No. 88072) using water. Peaks corresponding to dimethicone were detected by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector). Separation of the dimethicone from the hand lotion was not entirely successful with interfering sample components co-eluting with about half of the dimethicone. In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Edible Oil Adulteration by HPLC/ELSD
    Edible Oil Adulteration, Alltech Application Note 0056E, March 29, 2004. To detect adulteration of olive oil with vegetable oil by measuring triglycerides, a reversed phase HPLC procedure with ELSD detection was developed. Triglycerides in pure extra virgin olive oil, vegetable oil, a common adulterant of olive oils and a 50:50 mixture of the two oils were separated by gradient elution in around 15 minutes by reversed phase HPLC on an Alltima C18, 3m, 250 x 4.6mm column (Part No. 81387) using methylene choride/acetonitrile and were detected by evaporative light scattering using an Alltech Model 2000 (Evaporative Light Scattering Detector.) The fatty acids capric acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid (O), linoleic acid (L) and linolenic acid when combined together produce various triglycerides. The HPLC separations showed that the vegetable oil adulteration could be easily detected by the presence of the triglyceride trilinolein (LLL) that is significant in vegetable oil and the reduced presence of triolein (OOO) which is significant in olive oil. In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Egg Yolk Phospholipids by HPLC/ELSD
    Egg Yolk Phospholipids, Alltech Application Note 0008E, May 2, 2000. Egg yolk phospholipids were separated isocratically in around 20 minutes by reversed phase HPLC on a Nucleosil C18, 5m, 250 x 4.6mm column (Part No. 89141) using chloroform/acetonitrile and were detected by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector.) In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Egg Yolk Phospholipids by Normal Phase HPLC/ELSD
    Phospholipids- Normal Phase, Alltech Application Note 0024E, May 10, 2000. Egg yolk phospholipids and phospholipid standards were separated by normal phase HPLC/ELSD. Separation of a mixture of phospholipid standards was achieved by gradient elution with IPA/hexane/water in around 14 minutes using an Alltech Allsphere Silica Hypersil, 3m, 100 x 4.6mm column (Part No. 778382) and detection was by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector). Cholesterol, palmitic acid, phosphatidylethanolamine, phosphatidylserine, phosphatidylcholine and sphingomyelin were separated. Separation of egg yolk phospholipids was achieved by gradient elution with IPA/hexane/water in around 14 minutes using a Spherisorb Silica, 3m, 100 x 4.6mm column (Part No. 8382) and detection was by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector). Cholesterol, phosphatidylethanolamine, phosphatidylcholine and sphingomyelin were separated. In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Fat Soluble Vitamins by HPLC/ELSD
    Fat-Soluble Vitamins, Alltech Application Note 0007E, June 23, 2000. A mixture of five fat-soluble vitamins were separated isocratically in around 21 minutes by reversed phase HPLC on an Adsorbosphere C18, 5m, 250 x 4.6mm column (Part No. 287062) using methanol/acetonitrile after first dissolving them in chloroform. Vitamins A, D2, D3, E and K, were detected by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector.) In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Fatty Acid Methyl Esters by HPLC/ELSD
    Fatty Acid Methyl Esters, Alltech Application Note 0038E, July 10, 2000. The ELSD and UV detection of five fatty acid methyl esters (FAME) separated by reversed phase HPLC was compared. The FAME compounds were separated isocratically in around 10 minutes by reversed phase HPLC on a Silver Impregnated Ion-Exchange, 250 x 4.6mm column (Part No. not given) using acetonitrile/methanol and detected by evaporative light scattering (ELSD) using an Alltech Model 500 (Evaporative Light Scattering Detector) and by UV at 206 nm. All five of the FAME compounds (18:0, 18:1 n-9, 18:2, n-6, 18:3 n-3 and 20:4 n-6) were resolved by ELSD, but only the last 3 were resolved by UV. In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Fatty Acids by HPLC/ELSD
    Evaporative Light Scatting Detection Simplifies Fatty Acid Analysis, Alltech Application Note 0045E, June 5, 2000. Fatty acids cannot easily be determined by GC due to poor resolution and sample decomposition. Derivatization to allow their determination by reversed phase HPLC with UV detection is time consuming and complicated. HPLC separation of fatty acids with detection by evaporative light scattering does not require post-column derivatization as in the case of UV detection among other advantages. A mixture of six underivatized fatty acids (linolenic, myristic, linoleic, palmitic, oleic and stearic acid) ranging from 15-80mg/L was separated by gradient elution in around 20 minutes by reversed phase HPLC on an Alltima C18-LL, 5m, 250 x 2.1mm column (Part No. 88389) using water/acetonitrile and detected by evaporative light scattering using an Alltech Model 500 (Evaporative Light Scattering Detector.) The UV detection at 210 nn showed almost no peaks. A mixture of the same fatty acids at around 600mg/L separated using an analytical-bore column (Alltima C18-LL, 5m, 250 x 4.6mm, Part No. 88099) with a flow splitter produced better sensitivity than without one by allowing the drift tube temperature to be reduced thereby reducing sample evaporation. In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Galactose Standard by Reversed Phase HPLC/ELSD
    Galactose, Alltech Application Note 0043E, May 10, 2000. Galactose was separated isocratically in around 10 minutes by reversed phase HPLC on an Alltech 700 CH Carbohydrate, 300 x 6.5mm column (Part No. 70057) using water and detected by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector). A linear response was found with a detection limit of 10g/mL. In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Ginseng Root Compounds by HPLC/ELSD
    Analysis of Ginsenosides by HPLC with ELSD, Alltech Application Note 0051E, July 7, 2000. Ginseng root extracts, known as gensenosides and crude ginseng root powder were separated by gradient elution in around 20 minutes by reversed phase HPLC on a Partisil ODS-3, 5m, 250 x 4.6mm column (Part No. 8660) using water/acetonitrile. The ginsenosides Rg1 and Rb1 from a standard mixture and peaks corresponding to them in the crude root powder were detected by evaporative light scattering using an Alltech Model 500 (Evaporative Light Scattering Detector.) In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Glyceryl Monolaurate Purity by Reversed Phase HPLC/ELSD
    Monoglyceride Purity - Glyceryl Monolaurate, Alltech Application Note 0037E, April 21, 2000. Glyceryl monolaurate, a monoglyceride and its impurities were separated isocratically in around 10 minutes by reversed phase HPLC on a Nucleosil C18, 5m, 250 x 4.6mm column (Part No. 89141) using acetonitrile and detected by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector). In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • HPLC Determination of Organic Acids
    HPLC Determination of Organic Acids, Grom Application Number 10084 Oxalic acid, maleic acid, malonic acid, succinic acid, formic acid and acetic acid in a standard were separated using a GROM-RESIN AC organic acids HPLC column (300 x 8mm, Part No. GSPC11012S3008) by isocratic elution with 0.003N sulfuric acid in around 15 minutes and detected by conductivity.
  • Muscle Relaxant Drugs by HPLC/ELSD
    A mixure of four muscle relaxant drugs, rocuronium, pancuromium, pipecuronium and vecuronium bromide was separated by gradient elution in around 15 minutes by reversed phase HPLC on an Adsorbosphere UHS C18, 5m, 150 x 2.1mm column (Part No. 288435) using water/methanol. The drugs were detected by evaporative light scattering using an Alltech Model 500 (Evaporative Light Scattering Detector), by UV at 205 nm and by refractive index (RI). The ELSD baseline was more stable than that from the UV and RI determinations. The HPLC/ELSD detection limit was 9ng on column. In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Nandrolone by HPLC/ELSD
    Anabolic Steroids- Nandrolone, Alltech Application Note 0035E, June 5, 2000. Separation of the anabolic steroids nandrolone, 19-norepiandrosterone, 19-noretiocholanone, and 19-norandrosterone was achieved by reversed phase isocratic elution with acetonitile/water in around 6 minutes using an Adsorbosphere C18, 5m, 250 x 4.6mm column (Part No. 287062). Detection was by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector). In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Non Ionic Surfactant Tergitol NP-35 by HPLC/ELSD
    Non Ionic Surfactant Tergitol NP-35, Alltech Application Note 0030E, October 28, 1999. The non ionic surfactant Tergitol NP-35 was separated by gradient elution in around 60 minutes by normal phase HPLC on a Hypersil APS-2 (NH2), 5m, 250 x 4.6mm column (Part No. 11181) using hexane, IPA and water and detected by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector). In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Non-Ionic Detergent Glucopyranosides by HPLC/ELSD, UV
    Non-Ionic Detergent Glucopyranosides, Alltech Application Note 0010E, May 2, 2000. Glucopyranosides in non-ionic detergent were separated isocratically in around 5 minutes by reversed phase HPLC on an Econosphere C18, 5m, 150 x 4.6mm column (Part No. 70065) using methanol/water and were detected by UV at 220nm and by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector.) The UV detection did not resolve the individual glucopyranosides, while the ELSD produced distinct peaks for n-decyl B-D-glucopyranoside, n-octyl B-D-glucopyranoside and n-dodecyl B-D-glucopyranoside. In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Non-Ionic Surfactant Triton X 100 by HPLC/ELSD
    Non-ionic Surfactant Triton X 100, Alltech Application Note 0026E, July 7, 2000. To ensure the quality of commercial surfactant formulations, reliable techniques are needed to characterize components present in these products. HPLC is often used for this application. UV and refractive index (RI) detection methods generally dont work well in detecting surfactants due to lack of sufficient chromophores or unstable baselines. For these and other reasons, the evaporative light scattering detector can be used instead of UV or RI, since it detects any non-volatile analyte regardless of its optical properties. In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser. Separation of the surfactant Triton X 100 was achieved by reversed phase gradient elution with water/methanol in around 15 minutes using an Adsorbosphere XL TMS C1, 5m, 250 x 4.6mm column (Part No. 20628). Detection was by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector) with a Low Temperature Adaptor.
  • Nonphenol Ethoxylate Surfactant by HPLC/ELSD
    Surfactant Nonphenol Ethoxylate, Alltech Application Note 0039E, July 10, 2000. The surfactant nonphenol ethoxylate was separated by gradient elution in around 25 minutes by normal phase HPLC on a Supelco LC-DIOL, 250 x 4.6mm column (Part No. 88133) using hexane/methylene chloride and hexane/methanol and detected by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector). In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Pharmaceutical Stimulants by HPLC/ELSD
    Pharmaceutical Stimulants, Alltech Application Note 0049LE, July 7, 2000. Three pharmaceutical stimulants, theophylline, caffeine and ethamivan were separated by reversed phase HPLC/ELSD. A Low Temperature Adaptor was used to increase sensitivity and maintain stable baselines because a large portion of the mobile phase is aqueous and contains acetic acid. A mixture of the three stimulants was separated isocratically in around 5 minutes by reversed phase HPLC on a Platinum EPS C18, 5m, 250 x 4.6mm column (Part No. 32214) using methanol/acetonitrile/acetic acid. The stimulants were detected by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector). In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Polyethylene Glycol 600 by HPLC/ELSD
    Polymer-Polyethylene Glycol, Alltech Application Note 0029E, July 7, 2000. Polyethylene glycol MW 600 was separated by gradient elution in around 30 minutes by reversed phase HPLC on an Adsorbosphere C8, 5m, 250 x 4.6mm column (Part No. 287112) using water/methanol and detected by evaporative light scattering (ELSD) using an Alltech Model 500 (Evaporative Light Scattering Detector.). To achieve a good separation, the column and the tubing leading to it were heated with a preheating coil set at 70C. In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Polysaccharides Dextran T-10 and T-40 by HPLC/ELSD
    Polysaccharides, Alltech Application Note 0021E, May 2, 2000. The polysaccharides Dextran T-10 and T-40 were separated isocratically by reversed phase HPLC in around 10 minutes on an Alltech Macrosphere GPC 100, 7m, 250 x 4.6mm column (Part No. 88133) using water at pH 8.0 and detected by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector). In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Polystyrene MW Standards by HPLC/ELSD
    Polystyrene MW Standards, Alltech Application Note 0009E, July 7, 2000. Polystyrene molecular weight standards from 800 to 90,000 MW were separated isocratically in around 16 minutes by reversed phase HPLC on a Jordi Gel 500A, 500 x 10mm column (Part No. 100567) using 100% THF and were detected by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector.) In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Precision of n-Decyl beta-d Glucopyranoside by HPLC/ELSD
    Reproducibility- n-Decyl beta-d Glucopyranoside, Alltech Application Note 0022E, May 2, 2000. The variation in the area counts of the peaks from 11 separations of the surfactant n-Decyl beta-d Glucopyranoside by reversed phase HPLC and evaporative light scattering detection was determined to be less than 1%RSD. Separation was achieved isocratically with methanol/water by reversed phase HPLC in around 3 minutes using an Alltech Econosphere C18, 5m, 150 x 4.6mm column (Part No. 70065) and detection was by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector). In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Purity of Phospholipid Standards by Normal Phase HPLC/ELSD
    Phospholipid Class Standards- Normal Phase, Alltech Application Note 0036E, July 7, 2000. Five phospholipid standards were separated by normal phase HPLC/ELSD. Separation of a mixture of phospholipid standards was achieved by gradient elution with IPA/hexane/water/ammonium hydroxide in around 14 minutes using an Alltech Allsphere Silica Hypersil, 3m, 100 x 4.6mm column (Part No. 778382) with detection by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector). Phosphatidylinositol, phosphatidic acid, phosphatidylserine, phosphatidylcholine and sphingomyelin were separated. In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • SFC Steroid Analysis by HPLC/ELSD
    Supercritical Fluid Chromatography Steroid Analysis Using an ELSD, Alltech Application Note 0047LE, July 7, 2000. Modifiers and additives commonly used in supercritical fluid chromatography (SFC) interfere with FID or UV detection. An HPLC light scattering detector, the Alltech ELSD Mark III (Evaporative Light Scattering Detector) was modified to interface with an SFC system to evaluate performance of the ELSD in the reversed phase HPLC determination of steroids. A mixture of four steroids, progesterone, testosterone, 17-hydroxyprogesterone and hydrocortisone was separated separated by gradient elution in around 10 minutes by reversed phase HPLC on a Deltabond CN, 5m, 250 x 4.6mm column (Keystone Scientific, Inc) using methanol/carbon dioxide. The steroids were detected by evaporative light scattering using an Alltech ELSD Mark III (Evaporative Light Scattering Detector). Detection limits of 5-10ng were achieved. In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Steroid Conjugates by HPLC/ELSD
    Steroid Conjugates, Alltech Application Note 0042E, July 7, 2000. A mixure of three steroid conjugates, estrone 3-sulphate, b-estradiol 17-(b-D glucaronide) and 5b-pregnane-3a, 20a diol glucaronide (PdG) were separated by gradient elution in around 20 minutes by reversed phase HPLC on an Alltima C18, 5m, 150 x 2.1mm column (Part No. 88370) using water/methanol. The steroids were detected by evaporative light scattering using an Alltech ELSD Mark III (Evaporative Light Scattering Detector) and by UV at 210 nm. The ELSD baseline was more stable than that from the UV determination. The response factors for each were nearly the same except for PdG which was barely detected at all by UV due to its lack of sufficient chromophores for absorption. In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Steroids by HPLC/ELSD
    Steroids, Alltech Application Note 0027E, June 5, 2000. Four steroids (hydrocortisone, prednisone, testosterone and progesterone) were separated by HPLC/ELSD. Separation of the steroids hydrocortisone, prednisone, testosterone and progesterone was achieved by reversed phase isocratic elution with methanol/water in around 15 minutes using a Hypersil C18, 5m, 250 x 4.6mm column (Part No. 9879). Detection was by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector). In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Sugars in Food Products by HPLC/ELSD
    Carbohydrate Profiles-Representative Food Products, Alltech Application Note 0020E, March 29, 2004. Sugars in corn syrup and sugar alcohols used as sweeteners in food products were separated isocratically and by gradient elution by reversed phase HPLC on several different HPLC columns with acetonitrile/water and detected by evaporative light scattering using an Alltech 2000 ELSD (Evaporative Light Scattering Detector.) A mixture of sugars and sugar alcohols was separated by gradient elution in around 55 minutes on a Prevail Carbohydrate ES, 5m, 250 x 4.6mm column (Part No. 35101). Iso-erythritol, arabitol, fructose, sorbitol, mannitol, glucose, sucrose, maltitol, maltose, raffinose, maltotriose and stachyose were separated. Iso-erythritol, xylitol, mannitol and maltitol were separated isocratically in around 10 minutes on a Prevail Carbohydrate ES, 5m, 250 x 4.6mm column (Part No. 35101). Mannitol, glucose and sucrose in soy breakfast cereal were separated by gradient elution in 7 minutes on a Prevail Carbohydrate ES, 100 x 7mm Rocket column (Part No. 35103.) Mannitol, glucose, lactose and raffinose in an Asiago Cheese Bagel were separated by gradient elution in 5 minutes on a Prevail Carbohydrate ES, 100 x 7mm Rocket column (Part No. 35103.) Glucose, sucrose and lactose in a malted milk ball were separated by gradient elution in 7 minutes on a Prevail Carbohydrate ES, 100 x 7mm Rocket column (Part No. 35103.) Sorbitol, fructose, maltose, maltotriose, maltotetraose and maltopentaose in raspberry jam were separated by gradient elution in 7 minutes on a Prevail Carbohydrate ES, 100 x 7mm Rocket column (Part No. 35103.) Fructose, glucose and sucrose in clover honey were separated by gradient elution in around 10 minutes on a Prevail Carbohydrate ES, 5m, 250 x 4.6mm column (Part No. 35101).
  • Sugars in Fruit Juices by HPLC/ELSD
    Carbohydrate Profiles-Fruit Juice, Alltech Application Note 0053E, March 29, 2004. Simple sugars and sugar alcohols found in fruit juices were separated isocratically and by gradient elution by reversed phase HPLC on several different HPLC columns with acetonitrile/water and detected by evaporative light scattering using an Alltech 2000 ELSD (Evaporative Light Scattering Detector.) Fructose, glucose and sucrose in orange juice were separated isocratically in around 15 minutes on a Prevail Carbohydrate ES, 5m, 250 x 4.6mm column (Part No. 35101). Fructose, mannitol, glucose and sucrose in nectarine juice were separated by gradient elution in 7 minutes on a Prevail Carbohydrate ES, 100 x 7mm Rocket column (Part No. 35103.) Fructose, mannitol, glucose and sucrose and raffinose in plum juice were separated by gradient elution in 7 minutes on a Prevail Carbohydrate ES, 100 x 7mm Rocket column (Part No. 35103.) In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Surfactant DC-1503 Silicone Based Co-Polymer by HPLC/ELSD
    Surfactant DC-1503 Silicone Based Co-Polymer, Alltech Application Note 0018E, July 7, 2000. The surfactant DC-1503 silicone based co-polymer was separated isocratically in around 12 minutes by reversed phase HPLC on a Jordi Gel 500A, 500 x 10mm column (Part No. 100567) using 100% THF and detected by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector). In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Surfactant DC-190 Silicone Based Co-Polymer by HPLC/ELSD
    Surfactant DC-190 Silicone Based Co-Polymer, Alltech Application Note 0015E, July 7, 2000. The surfactant DC-190 silicone based co-polymer was separated isocratically in around 12 minutes by reversed phase HPLC on a Jordi Gel 500A, 500 x 10mm column (Part No. 100567) using 100% THF and detected by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector). In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Surfactant DC-193 Silicone Based Co-Polymer by HPLC/ELSD
    Surfactant DC-193 Silicone Based Co-Polymer, Alltech Application Note 0016E, July 7, 2000. The surfactant DC-193 silicone based co-polymer was separated isocratically in around 12 minutes by reversed phase HPLC on a Jordi Gel 500A, 500 x 10mm column (Part No. 100567) using 100% THF and detected by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector). In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Surfactant DC-194 Silicone Based Co-Polymer by HPLC/ELSD
    Surfactant DC-194 Silicone Based Co-Polymer, Alltech Application Note 0015E, July 7, 2000. The surfactant DC-194 silicone based co-polymer was separated isocratically in around 12 minutes by reversed phase HPLC on a Jordi Gel 500A, 500 x 10mm column (Part No. 100567) using 100% THF and detected by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector). In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Surfactant Plurafac B-26 by HPLC/ELSD
    Surfactant Plurafac B-26, Alltech Application Note 0014E, July 7, 2000. The surfactant Plurafac B-26 was separated isocratically in around 12 minutes by reversed phase HPLC on a Jordi Gel 500A, 500 x 10mm column (Part No. 100567) using 100% THF and detected by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector). In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Surfactant Pluronic L-62 by HPLC/ELSD
    Surfactant Pluronic L-62, Alltech Application Note 0013E, July 7, 2000. The surfactant Pluronic L-62 was separated isocratically in around 12 minutes by reversed phase HPLC on a Jordi Gel 500A, 500 x 10mm column (Part No. 100567) using 100% THF and detected by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector). In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Testosterone by HPLC/ELSD
    Anabolic Steroids- Testosterone and Epitesterone, Alltech Application Note 0033E, June 23, 2000. Separation of the anabolic steroids testosterone and epitesterone was achieved by reversed phase isocratic elution with acetonitile/water in around 6 minutes using an Adsorbosphere C8, 5m, 250 x 4.6mm column (Part No. 287112). Detection was by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector). In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser. See also chromatogram 7407.
  • Triglycerides in Lip Balm by HPLC/ELSD
    Triglycerides in Chapstick, Alltech Application Note 0005E, April 24, 2000. Triglycerides in a commercial lip balm, Chapstick, were separated in around 10 minutes by reversed phase HPLC on a Hypersil C18, 5m, 250 x 4.6mm column (Part No. 9879) using gradient elution with acetone and ethanol. The compounds were detected by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector.) In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Triglycerides in Olive Oil by HPLC/ELSD
    Triglycerides Olive Oil, Alltech Application Note 0003E, May 8, 2000. Triglycerides in olive oil were separated by reversed phase HPLC on a Hypersil C18, 5m, 250 x 4.6mm column (Part No. 9879) using gradient elution with acetonitrile and methanol. UV detection at 225 nm showed almost no discernible peaks, while detection by evaporative light scattering detection (ELSD) using an Alltech 500 ELSD (Evaporative Light Scattering Detector) found more than a dozen individual components over a 40-minute period. In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Underivatized Amino Acids by HPLC/ELSD
    Underivatized Amino Acids, Alltech Application Note 0040E, July 10, 2000. A mixture of twelve underivatized amino acids was separated by gradient elution in around 20 minutes by reversed phase HPLC on an Alltima C18, 5m, 250 x 4.6mm column (Part No. 88056) using water/acetonitrile. The amino acids, serine, lysine, glutamic acid, arginine, proline, valine, methionine, tyrosine, isoleucine, leucine, phenylalanine and tryptophan were detected by evaporative light scattering using an Alltech Model 500 (Evaporative Light Scattering Detector.) The HPLC/ELSD detection limits ranged from 9 to 328ng on column. In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Underivatized C14-C18 Fatty Acids by HPLC/ELSD
    Fatty Acids (Underivatized), Alltech Application Note 0025E, June 5, 2000. Separation of a mixture of six underivatized fatty acid standards was achieved by gradient elution with water/acetonitrile in around 20 minutes using an Alltech Alltima C18-LL, 5m, 250 x 2.1mm column (Part No. 88389). Detection was by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector). Linolenic acid, myristic acid, linoleic acid, palmitic acid, oleic acid and stearic acid were separated. In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.
  • Water Soluble Vitamins by HPLC/ELSD
    Water-Soluble Vitamins, Alltech Application Note 0006E, June 5, 2000. A mixture of four water-soluble vitamins was separated in around 17 minutes by reversed phase HPLC on an Alltima C18, 5m, 250 x 2.1mm column (Part No. 88371) using gradient elution with ammonium acetate in water and acetonitrile. The vitamins, ascorbic acid, niacin, thiamine and riboflavin were detected by evaporative light scattering using an Alltech 500 ELSD (Evaporative Light Scattering Detector.) In ELSD, the mobile phase is first evaporated. Solid particles remaining from the sample are then carried in the form of a mist into a cell where they are detected by a laser.